U.S. patent application number 16/315278 was filed with the patent office on 2019-12-05 for diagnosing col6-related disorders and methods for treating same.
The applicant listed for this patent is THE GENERAL HOSPITAL CORPORATION, MURDOCH UNIVERSITY, PRESIDENT AND FELLOWS OF HARVARD COLLEGE, THE USA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES, UCL BUSINESS PLC, THE USA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES. Invention is credited to Veronique BOLDUC, Carsten G. BONNEMANN, Beryl CUMMINGS, Monkol LEK, Daniel MACARTHUR, Francesco MUNTONI, Steve WILTON.
Application Number | 20190367917 16/315278 |
Document ID | / |
Family ID | 60901397 |
Filed Date | 2019-12-05 |
View All Diagrams
United States Patent
Application |
20190367917 |
Kind Code |
A1 |
BONNEMANN; Carsten G. ; et
al. |
December 5, 2019 |
DIAGNOSING COL6-RELATED DISORDERS AND METHODS FOR TREATING SAME
Abstract
A single nucleotide polymorphism (SNP) that results in
development of a Type VI collagen, alpha 1 chain-related disorder,
and the use of the SNP to identify individuals at risk for
developing COL6-related disorders (COL6-RD). Also provided are
antisense oligomers for treating individuals at risk for developing
COL6-RD, as well as methods for screening compounds for their
potential as therapeutic agents.
Inventors: |
BONNEMANN; Carsten G.;
(Washington, DC) ; BOLDUC; Veronique; (Rockville,
MD) ; MUNTONI; Francesco; (London, GB) ;
WILTON; Steve; (Applecross, Western Australia, AU) ;
MACARTHUR; Daniel; (Cambridge, MA) ; LEK; Monkol;
(Braintree, MA) ; CUMMINGS; Beryl; (Brookline,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE USA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN
SERVICES
MURDOCH UNIVERSITY
THE GENERAL HOSPITAL CORPORATION
PRESIDENT AND FELLOWS OF HARVARD COLLEGE
UCL BUSINESS PLC |
Bethesda
Perth, Western Australia
Boston
Cambridge
London |
MD
MA
MA |
US
AU
US
US
GB |
|
|
Family ID: |
60901397 |
Appl. No.: |
16/315278 |
Filed: |
July 5, 2017 |
PCT Filed: |
July 5, 2017 |
PCT NO: |
PCT/US2017/040726 |
371 Date: |
January 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62358482 |
Jul 5, 2016 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12N 2800/60 20130101; C12Q 1/6886 20130101; C12Q 1/6883 20130101;
C12N 15/113 20130101; C12N 15/86 20130101; C12N 2310/3233 20130101;
C12N 2320/33 20130101; C12N 15/111 20130101; C12N 2310/11
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; C12Q 1/6883 20060101 C12Q001/6883; C12N 15/86 20060101
C12N015/86 |
Claims
1. An antisense oligomer targeted to a sequence in intron 11 of
COL6A1 pre-mRNA molecule, wherein the oligomer has been modified to
reduce degradation of the oligomer, and wherein hybridization of
the antisense oligomer to the target sequence in the COL6A1
pre-mRNA molecule having a non-native splice donor or splice
acceptor site, results in normal splicing of the COL6A1 pre-mRNA
molecule having the non-native splice donor or splice acceptor
site.
2. The antisense oligomer of claim 1, wherein hybridization of the
antisense oligomer to the target sequence results in production of
a mature COL6A1 mRNA lacking SEQ ID NO:4.
3. The antisense oligomer of claim 1, wherein the non-native
splice-donor or splice acceptor site is located within intron
11.
4. The antisense oligomer of claim 1, wherein the target sequence
is at least 90% identical to a polynucleotide sequence in SEQ ID
NO:3.
5. The antisense oligomer of claim 1, wherein the antisense
oligomer is sufficiently complementary to the target sequence such
that the oligomer specifically hybridizes to the target
sequence.
6. The antisense oligomer of claim 1, wherein the target sequence
comprises a sequence at least 90% identical to a sequence selected
from the group consisting of SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ
ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, and SEQ ID
NO:58.
7. The antisense oligomer of claim 1, wherein the antisense
oligomer comprises a sequence at least 95% identical to a sequence
selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16,
SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
NO:43, and SEQ ID NO:44.
8. The antisense oligomer of claim 1, wherein the antisense
oligomer is an antisense RNA molecule.
9. A method of modulating splicing of a COL6A1 pre-mRNA molecule
having a non-native splice donor or splice acceptor site, the
method comprising contacting a cell expressing the COL6A1 pre-mRNA
molecule having the non-native splice donor or splice acceptor
site, with the antisense oligomer of claim 1.
10. A method of treating a collagen VI-related disorder (COLVI-RD),
comprising administering to an individual in need of such treatment
the antisense oligomer of claim 1.
11. An expression vector encoding the antisense oligomer of claim
1.
12. A method of identifying a patient at risk for developing a
neuromuscular disorder, comprising, analyzing a sample from a
patient to determine if the patient has one or more mutations in an
intronic sequence of the patient's COL6A gene, wherein the one or
more mutations create a non-native splice donor or splice acceptor
site; wherein if the one or more mutations are detected,
identifying the patient as being at risk for developing a
neuromuscular disorder.
13. (canceled)
14. The method of claim 12, wherein the non-native splice donor or
splice acceptor site is in intron 11 of the COL6A gene.
15. The method of claim 12, wherein the mutation is present at a
genomic position corresponding to position 21 of SEQ ID NO:6.
16. (canceled)
17. A recombinant nucleic acid molecule comprising an insert, the
insert comprising at least a portion of an intron from a COL6A gene
flanked by at least a 3' splice acceptor site and at least a 5'
splice donor site, wherein the intron portion comprises a mutation
that creates a non-native splice donor or splice acceptor site in
the portion.
18. The recombinant expression vector of claim 17, wherein the
intron is intron 11 of the COL6A gene.
19. The recombinant expression vector of claim 17, wherein the
intron comprises SEQ ID NO:6.
20. The recombinant expression vector of claim 17, wherein the
intron comprises SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.
21. The recombinant expression vector of claim 17, wherein the
splice donor site is from exon 11 of a COL6A gene, and wherein the
splice acceptor site is from exon 12 of a COL6A gene.
22. A method of identifying compounds capable of modulating
splicing of a COL6A1 pre-mRNA, comprising: contacting a test
compound with a cell comprising a recombinant expression vector of
claim 17; and, determining if mRNA transcribed from the recombinant
expression vector comprises the intron portion; wherein if mRNA
transcribed from the recombinant nucleic acid molecule lacks the
intron portion, identifying the test compound as capable of
modulating splicing of COL6A1 pre-mRNA.
Description
REFERENCE TO SEQUENCE LISTING
[0001] This application contains a Sequence Listing submitted as an
electronic text file named
"6137NINDS-2-PCT_sequence_listing_ST25.txt", having a size in bytes
of 34 KB, and created on Jul. 5, 2017. The information contained in
this electronic file is hereby incorporated by reference in its
entirety pursuant to 37 CFR .sctn. 1.52(e)(5).
TECHNOLOGICAL FIELD
[0002] This disclosure relates to identifying individuals at risk
of developing, or who have, a COL6A1-related disorder such as
Ullrich muscular dystrophy, by detecting a specific nucleotide
substitution in the gene encoding the alpha 1 chain of Type VI
collagen. It also relates to the use of antisense oligonucleotides
for treating individuals having such nucleotide substitution.
BACKGROUND
[0003] Collagen type VI is an important component of the
extracellular matrix and plays crucial roles in organizing the
matrix, and in supporting cell adhesion and survival. Type VI
collagen is abundant predominantly in muscle, tendon and skin,
tissues where collagen VI dysfunction may have important clinical
sequelae. Collagen VI is produced from three independent genes,
COL6A1, COL6A2 and COL6A3, each encoding an alpha chain essential
for forming the collagen VI monomer, the basic unit of this complex
protein. Three additional collagen VI genes have been identified in
recent years (COL6A4, COL6A5, and COL6A6), although their
importance is unknown at this time. The three alpha chains
(.alpha.1, .alpha.2, and .alpha.3), which share a similar
structure, namely a triple helical (TH) domain composed of the
Gly-X-Y repeated motif flanked by two globular domains, come
together starting at the C-terminal end of the TH domain, and
assemble, in a zipper-like fashion, through hydrogen bonding
mediated by the glycine residues. Two monomers assemble to form
antiparallel dimers, and subsequently tetramers, and these
quaternary structures are stabilized by disulfur bridges from
critical cysteine residues present in the TH domains. Tetramers are
secreted in the extracellular space, where they unite end-to-end to
form microfibrils. Residues in globular domains of the three chains
are important for these interactions. In muscle, collagen VI
microfibrils are located at the interface of the extracellular
matrix and the myofibers basement membrane, from where they bind to
other constituents of these networks, such as collagen type IV and
biglycan. The main sources of collagen VI in muscle are the
interstitial fibroblasts, as opposed to the muscle fibers
themselves.
[0004] Mutations in any of the three main collagen VI genes
(COL6A1, COL6A2 and COL6A3) are responsible for a number of
neuromuscular disorders, collectively referred as collagen
VI-related disorders (COL6-RD), that are now considered part of a
spectrum rather than as distinct disorders. Ullrich muscular
dystrophy, on the severe end of the spectrum, manifests by
progressive, early-onset muscle weakness, proximal joint
contractures, distal joint hyperlaxity, and respiratory
dysfunction. Ullrich patients never acquire the ability to walk, or
are delayed and later lose ambulation. Respiratory insufficiency is
also a critical aspect of the disease, as it can be
life-threatening if not properly managed. Bethlem myopathy, which
is on the mild end of the spectrum, also presents with muscle
weakness, proximal joint contractures, and distal laxity, although
moderate. It is an adult-onset disorder, usually not associated
with loss of ambulation. In between Ullrich and Bethlem myopathy
lie a series of intermediate phenotypes with different degrees of
disease severity.
[0005] Col6-RD can be inherited as recessive or dominant, but are
most commonly caused by de novo dominant-negative mutations that
act by interfering at different stages of the assembly process of
the collagen VI tetramer molecules. Mutations that prevent mutant
monomers from assembling into dimers and tetramers should be
associated with a milder phenotype, as only normal tetramers are
secreted. Alternatively, mutations that can be carried up until the
tetramers will have a strong dominant-negative effect, as the vast
majority of tetramers will be dysfunctional in the extracellular
space. The two main categories of dominant-negative mutations are
glycine substitutions (in the Gly-X-Y motif of the TH domain), and
in-frame exon deletions (or mutations resulting in in-frame exon
skipping), occurring typically at the N-terminal end of the TH, and
therefore being incorporated into monomers. These mutations usually
result in mislocalization of collagen VI in the muscle tissue, and
reduced collagen VI deposition and increased retention in the
cultured fibroblasts.
[0006] Patients suffering from COL6-RD usually present with
progressive muscle weakness and stiffness in the spine and joints.
Following clinical assessment, a suspicion of COL6-RD can be
confirmed in the laboratory through standard diagnosis tools
including biochemical analyses (muscle biopsy and cultured
fibroblasts immunostaining) and genetic testing. Targeted genetic
testing by sequencing the cDNA of the triple helical domains of
COL6A1, COL6A2 and COL6A3 efficiently detects most of the
mutations, such as exon deletion and glycine substitution
mutations. Despite the availability of such procedures, they fail
to identify a mutational cause for a considerable number of
patients who meet the clinical and biochemical criteria of COL6-RD.
Such findings suggest that additional mutations in collagen VI, or
other genes, exist and remain to be identified and/or associated
with COL6-RD.
[0007] Currently no cure exists for COL6-RD. The main form of
treatment is physiotherapy, the goal of which is to keep the
muscles active and to prevent the formation of contractures. If
scoliosis develops, a spine brace may help prevent further
deterioration, and severe cases may need surgical correction.
Finally, night-time breathing problems may occur, resulting in
headaches, drowsiness, and loss of appetite and weight, and which
requires the initiation of night time mechanical ventilatory
support. Respiratory failure can then progress to require daytime
mechanical ventilatory support.
[0008] It is clear that current methods of diagnosing and treating
COL6-RD are insufficient. Moreover, the currently available
treatments merely attempt to slow onset or worsening of the
disease, and fail to provide a permanent cure. Thus, what is needed
are improved methods for diagnosing and treating individuals at
risk for, or who are suffering from, COL6-RD. The present
application provides such methods and therapies, and offers other
benefits as well.
SUMMARY
[0009] The inventors have discovered a mutation in intron 11 of the
COL6A1 gene that alters splicing of COL6A1 pre-mRNA, and produces a
mature alpha 1(VI) chain mRNA that comprises an additional exon.
Translation of such mRNA results in the production of an aberrant
Type VI alpha 1 chain protein, leading to the development of
neuromuscular disorders. Detection of this mutation can be used to
diagnose individuals at risk for developing collagen VI-related
disorders. Normal splicing of pre-mRNA containing the mutation can
be achieved through the use of exon-skipping technology.
[0010] Thus, this disclosure provides antisense oligomers targeted
to a sequence in intron 11 of an COL6A1 pre-mRNA molecule, wherein
hybridization of the antisense oligomer to the target sequence in
mutated COL6A1 pre-mRNA results in production of normal (wt) alpha
1(VI) chain protein. In one aspect, hybridization of the antisense
oligomer to the target sequence results in normal splicing from
exon 11 to exon 12. In one aspect, production of a normal alpha
1(VI) chain protein is due to the alteration or modulation of
splicing resulting from hybridization of the antisense oligomer to
the target sequence in the pre-mRNA. In one aspect, hybridization
of the antisense oligomer to the target sequence results in
production of a mature COL6A1 mRNA lacking a pseudo-exon (SEQ ID
NO:4). In one aspect, hybridization of the antisense oligomer to
the target sequence results in production of a mature alpha 1(VI)
chain protein lacking SEQ ID NO:5. In one aspect, hybridization of
the antisense oligomer to the target sequence results in production
of a mature COL6A1 mRNA encoding a normal alpha 1(VI) chain
protein. In one aspect, hybridization of the antisense oligomer to
the target sequence results in production of a mature COL6A1 mRNA
comprising SEQ ID NO:61. In one aspect, hybridization of the
antisense oligomer to the target sequence results in production of
a mature COL6A1 mRNA encoding a protein comprising SEQ ID
NO:62.
[0011] Antisense oligomers of this disclosure may specifically
hybridize with polynucleotide sequences in intron 11 of COL6A1
pre-mRNA. In one aspect, the antisense oligomer does not hybridize
with sequences in exon 11 or exon 12. In one aspect, the target
sequence is at least 90% identical to a polynucleotide sequence in
intron 11 of COL6A1 pre-mRNA. In one aspect, the target sequence is
at least 90% identical to a polynucleotide sequence in SEQ ID NO:3.
In one aspect, the antisense oligomer is targeted to a
polynucleotide sequence in SEQ ID NO:3 or SEQ ID NO:4.
[0012] The length of antisense oligomers of the invention can be
optimized for specific hybridization to a target sequence. In one
aspect, the antisense oligomer is 10 to 50 nucleotides in length.
In one aspect, the antisense oligomer is 10 to 30 nucleotides in
length. In one aspect, the antisense oligomer is 15 to 25
nucleotides in length. In one aspect, the antisense oligomer
comprises, or consists of, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50
nucleobases.
[0013] Preferred antisense oligomers are those having a high degree
of complementarity with the target sequence. In one aspect, the
antisense oligomer is sufficiently complementary to the target
sequence such that the antisense oligomer specifically hybridizes
to a COL6A1 pre-mRNA comprising the target sequence. In one aspect,
the antisense oligomer comprises a nucleic acid sequence comprising
at least six contiguous nucleobases fully complementary to at least
six contiguous nucleobases in the target sequence. In one aspect,
the target sequence comprises a sequence at least 90% identical to
a SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, NO:26, SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID
NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ
ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,
SEQ ID NO:56, SEQ ID NO:57, or SEQ ID NO:58. In one aspect, the
target sequence comprises SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,
NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ
ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,
SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID
NO:58.
[0014] In one aspect, the antisense oligomer comprises a sequence
at least 85%, at least 90%, at least 95%, at least 97% or at least
99% identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44.
In one aspect, the antisense oligomer comprises SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, or SEQ ID NO:44.
[0015] Antisense oligomers can be made from RNA, DNA, combinations
thereof, and/or modified forms thereof. In one aspect, the
antisense oligomer is an antisense RNA molecule. In one aspect, the
antisense oligomer is an RNA molecule that comprises a modification
selected from the group consisting of a nucleoside modification, an
internucleoside modification, a sugar modification, a
sugar-internucleoside linkage modification, a peptide addition, and
combinations thereof. In one aspect, the antisense oligomer is
modified to reduce degradation by a ribonuclease. The antisense
oligomer may be a morpholino oligomer.
[0016] Antisense oligomers of this disclosure may be produced using
expression vectors. One aspect of the invention is an expression
vector encoding an antisense oligomer of the invention. In one
aspect, the expression vector is an isolated nucleic acid molecule.
In one aspect, the expression vector is viral expression vector
(e.g., AAV vector).
[0017] Antisense oligomers of the invention can be used to modulate
splicing of COL6A1 pre-mRNA. One aspect of the invention is a
method of modulating splicing of a COL6A1 pre-mRNA molecule
comprising a non-native splice donor or splice acceptor site in
intron 11, comprising contacting a cell expressing COL6A1 pre-mRNA
molecule comprising a non-native splice donor or splice acceptor
site with an antisense oligomer of the invention or an expression
vector expressing an oligomer of the invention.
[0018] One aspect of the invention is a method of treating a
collagen VI-related disorder (COL6-RD), comprising administering to
an individual in need of such treatment an antisense oligomer of
this disclosure. In one aspect, the COL6-RD is a COL6A1-RD. In one
aspect, the individual comprises a COL6A1 gene comprising a
mutation in intron 11 that introduces a new splice donor site. In
one aspect, the individual comprises a COL6A1 gene comprising a
NH_001848 c.930+189 C>T mutation. In one aspect, the individual
is treated by administering to the individual an expression vector
expressing an antisense oligomer of this disclosure. In one aspect,
the individual is treated by administering to the individual an
antisense oligomer targeted to a sequence comprising SEQ ID NO:3 or
SEQ ID NO:4. In one aspect, the individual is treated by
administering to the individual an antisense oligomer targeted to a
sequence comprising a sequence at least 90% identical to SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, or SEQ ID NO:58. In one aspect, the individual
is treated by administering to the individual an antisense oligomer
targeted to a sequence comprising SEQ ID NO:23, SEQ ID NO:24, SEQ
ID NO:25, NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ
ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, or SEQ ID
NO:58.
[0019] In one aspect, the individual is treated by administering to
the individual an antisense oligomer comprising a sequence at least
85%, at least 90%, at least 95%, at least 97%, or at least 99%
identical to SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44.
In one aspect, the individual is treated by administering to the
individual an antisense oligomer comprising SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,
SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID
NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ
ID NO:43, or SEQ ID NO:44.
[0020] One aspect of the invention is a method of diagnosing the
likelihood of an individual to develop a Collagen VI related
disorder (COL6-RD) comprising obtaining a biological sample from
the individual, and analyzing the sample to determine if the C or T
allele is present at a locus in chromosome 21 represented by SEQ ID
NO:3 or SEQ ID NO:6, wherein the presence of the T allele indicates
the individual will develop a COL6-RD.
[0021] One aspect of the invention is a method of diagnosing the
likelihood of an individual to develop a neuromuscular disorder,
comprising obtaining a biological sample from the individual, and,
analyzing the sample to determine if the C or T allele is present
at a locus in chromosome 21 represented by SEQ ID NO:3 or SEQ ID
NO:6, wherein the presence of the T allele indicates the individual
will develop a neuromuscular disorder.
[0022] One aspect of the invention is a method of diagnosing the
likelihood of an individual to develop Ullrich muscular dystrophy,
comprising obtaining a biological sample from the individual, and,
analyzing the sample to determine if the C or T allele is present
at a locus in chromosome 21 represented by SEQ ID NO:3 or SEQ ID
NO:6, wherein the presence of the T allele indicates the individual
will develop Ullrich muscular dystrophy.
[0023] One aspect of the invention is a method of diagnosing a
COL6-RD in an individual comprising: [0024] a. obtaining a plasma
sample from an individual, [0025] b. separating the sample into a
cellular and non-cellular fraction, [0026] c. detecting the
presence of the T allele in the cellular fraction, and [0027] d.
providing a diagnosis of a COL6-RD based on the presence of the T
allele in the cellular fraction of the sample.
[0028] In these methods, the biological sample may comprise a blood
sample, a tissue sample (esp. a muscle biopsy), and a buccal swab.
The presence or absence of the C or T allele may be detected by
analyzing genomic DNA, RNA transcripts, and/or the alpha1(VI)
protein.
[0029] Another aspect of this disclosure is a method of detecting a
SNP in intron 11 of the COL6A1 gene, comprising obtaining a nucleic
acid sample from an individual that includes a locus in chromosome
21 represented by SEQ ID NO:3, and detecting the presence of the T
allele at position 21 of SEQ ID NO:6.
[0030] Another aspect of this disclosure is a method of detecting a
SNP in intron 11 of the COL6A1 gene, comprising obtaining a nucleic
acid sample from an individual that includes a locus in chromosome
21 represented by SEQ ID NO:3, and detecting the presence of the T
allele at position 21 of SEQ ID NO:6.
[0031] Another aspect of this disclosure is a method of detecting a
SNP in intron 11 of the COL6A1 gene, comprising obtaining a plasma
sample from a human patient, and detecting whether the T allele or
the C allele is present at position 21 of the genomic DNA sequence
of SEQ ID NO:6.
[0032] Another aspect of this disclosure is a method of detecting
Ullrich muscular dystrophy in a patient comprising obtaining a
plasma sample from a human patient, and detecting whether the T
allele or the C allele is present at position 21 of the genomic DNA
sequence of SEQ ID NO:6.
[0033] Another aspect of this disclosure is a method of confirming
a diagnosis of Ullrich muscular dystrophy in a patient comprising
obtaining a plasma sample from a human patient, and detecting
whether the T allele or the C allele is present at position 21 of
the genomic DNA sequence of SEQ ID NO:6, wherein the presence of
the T allele is confirmatory of a diagnosis of Ullrich muscular
dystrophy.
[0034] This disclosure also provides recombinant nucleic acid
molecules comprising an insert comprising at least a portion of
intron 11 comprising SEQ ID NO:6, wherein the portion of intron 11
is flanked by at least a 5' splice donor site and at least a 3'
splice acceptor site, the insert being operationally linked to a
promoter sequence.
[0035] Another aspect of this disclosure is a method of identifying
compounds capable of modulating splicing of COL6A1 pre-mRNA,
comprising: introducing a test compound into a cell comprising a
recombinant expression vector comprising an insert comprising at
least a portion of intron 11 comprising SEQ ID NO:6, wherein the
portion of intron 11 is flanked by at least a 5' splice donor site
and at least a 3' splice acceptor site, the insert being
operationally linked to a promoter sequence; performing a first
polymerase chain reaction (PCR) assay on nucleic acid molecules
obtained from the cell, using a set of primers that bind sequences
flanking the 3' splice acceptor-intron 11 portion-5' splice donor
insert; and, comparing the size of the PCR product with a PCR
product produced from a second PCR assay performed on a second cell
comprising the recombinant expression vector but lacking the test
antisense oligomer, and using the same pair of primers; wherein if
the PCR product produced from the first PCT assay is smaller than
the PCR product produced in the second PCR assay, identifying the
test compound as capable of modulating splicing of COL6A1
pre-mRNA.
[0036] This disclosure also provides kits for practicing methods of
the invention. These kits may be used to predict the risk of an
individual to develop a COL6-RD, and may comprise an antisense
oligomer of the invention; and instructions for using antisense
oligomer. In one aspect, the kit is useful for modulating splicing
of COL6A1 pre-mRNA, and comprises an antisense oligomer of the
invention and instructions for using the antisense oligomer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows RNA-sequencing analysis (top) of two collagen
VI patients revealed the retention of intronic sequence (72 bp) in
mature transcripts between exon 11 and exon 12 of COL6A1. This
splicing event was not identified in 195 additional patient and
control samples. Whole-genome sequencing of patient 1 (bottom)
showed the presence of a de novo heterozygous mutation in intron
11, adjacent to the new splicing event.
[0038] FIG. 2 shows confirmation that the mutation correlates with
insertion of a 72 bp, intronic sequence into the mature mRNA. The
pseudo-exon inclusion was detected with reverse transcriptase
(RT)-PCR in patient-derived skin fibroblasts and muscle biopsy, and
showed low abundance as compared to the normal allele.
[0039] FIG. 3 shows the frequency in the NIH cohort of known
mutations in COL6 genes, as well as the COL6A1 intronic mutation of
this disclosure. In the NIH cohort, the COL6A1 intronic mutation
was the most common mutation.
[0040] FIG. 4 is an illustration of the reporter constructs
("minigene" constructs) prepared from portions of the COL6A1
genomic DNA sequence.
[0041] FIG. 5 shows an analysis of the effect of the T allele on
splicing from the reporter constructs illustrated in FIG. 4.
Expressing the splicing reporters in HEK293T cells demonstrated
aberrant splicing only on the reporter constructs carrying the
mutant ("T") allele.
[0042] FIG. 6 illustrates the therapeutic approach of using splice
modulation to "skip" the mutant exon in the COL6A1 gene in patients
with the T allele.
[0043] FIGS. 7A-C shows the strategy for design and testing of
2'OMe antisense oligonucleotides (AON). FIG. 7A illustrates the
design and positioning strategy of the tested AONs. FIG. 7B shows
representative gel images of HEK293T cells transfected with the
minigene construct (+Ex-11-13), and treated with the 2'OMe
antisense oligonucleotides, performed in duplicate and amplified by
reverse-transcriptase PCR. FIG. 7C shows the percentage of
expression determined using the gel density to measure the ratio of
mutant to normal expression of the experiments illustrated in FIG.
7B.
[0044] FIGS. 8A-8C. FIG. 8A shows the strategy used to design
phosphorothioate morpholino antisense oligonucleotides (PMO) to
promote skipping of the pseudo-exon. The PMO were positioned either
at the splice acceptor site (PMO-1, PMO-1b, PMO-1c), at the splice
donor site (PMO-3, PMO-3b, PMO-3c), or within the pseudo-exon at a
predicted splicing enhancer site (PMO-2, PMO-2b, PMO-2c, PMO-2d,
PMO-2e, PMO-2f, PMO-4, PMO-5). FIG. 8B shows representative gel
images of HEK cells transfected with the minigene construct
(+Ex-11-13), and treated with the PMO antisense oligonucleotides,
performed in duplicate and amplified by reverse-transcriptase PCR.
FIG. 8C shows the percentage of expression determined using the gel
density to measure the ratio of mutant to normal expression of the
experiments illustrated in FIG. 8B.
[0045] FIGS. 9A-9D. FIG. 9A shows the relative pseudoexon
expression levels in patient-derived cultured fibroblasts treated
with the indicated oligomer. A reverse-transcriptase PCR assay
designed to specifically detect the pseudoexon was used to measure
its expression. Bars represent the average of three biological
replicates.+-.standard error of the mean. Each biological replicate
is the average of 2 to 3 transfections. Statistical analyzes were
performed using multiple comparisons ANOVA followed by Dunnet
correction, for each treatment compared to PMO-Negative treatment.
*p<0.01, **p<0.001. FIG. 9B shows the relative pseudoexon
expression levels (calculated as in FIG. 9A) in fibroblasts treated
with the indicated combination of oligomers. FIG. 9C shows
representative images of patient-derived fibroblasts treated with
either a non-targeting PMO (PMO-Neg), or with a combination of
PMO-2b and PMO-3 for 5 days, and with sodium ascorbate for 3 days,
before staining for matrix-deposited collagen VI. FIG. 9D shows the
number of tetramers per microfibril following treatment with PMO-2b
and PMO-3, calculated using rotary shadowing electron microscopy
images.
[0046] FIGS. 10A and B. FIG. 10A is a schematic a chimeric splicing
reporter that was prepared by cloning the mouse genomic sequence
encompassing exon 11 to exon 13, and by replacing the intron 11
sequence with human intron 11, in presence of the wildtype (+189C)
or the mutant (+189T) genotype. FIG. 10B shows the results of
assays in which reporter constructs were transfected in murine
primary skin fibroblasts, and expression from the splicing reporter
analyzed by reverse transcriptase PCR (RT-PCR).
[0047] FIG. 11 is a schematic of the CRISPR/Cas9 strategy to
replace mouse sequence spanning exon 9 to exon 14 with its human
counterpart. The donor template will be synthesized in two
versions: one carrying the normal allele (+189C), one carrying the
mutant allele (+189T), to generate two humanized alleles.
DETAILED DESCRIPTION
[0048] This disclosure provides methods of identifying individuals
at risk for developing Collagen VI-related disorders (COL6-RD), as
well as methods for treating such individuals. The invention is
based on the inventors' discovery of a mutation in the COL6A1 gene
(NCBI Gene ID: 1291) that results in production of a mutant form of
the Type VI collagen alpha 1 chain protein ("alpha 1(VI) chain"),
leading to neuromuscular disorders. The newly discovered
substitution mutation (C>T) at a specific location in the region
between exon 11 and exon 12 of the alpha 1(VI) chain gene (COL6A1)
introduces a new, functional 5'-splice donor site in the pre-mRNA
molecule transcribed from the mutated gene. Utilization of the
splice donor site resulting from this mutation alters splicing of
COL6A1 pre-mRNA, such that the mature mRNA contains 72 additional
nucleotides (a pseudo-exon; SEQ ID NO:4) between exon 11 and exon
12. Individuals possessing the T allele of this C>T mutation
that results in the inclusion of the pseudo-exon in the COL6A1 gene
transcripts develop COL6-RD, particularly neuromuscular disorders
such as Ullrich muscular dystrophy.
[0049] The inventors tested the frequency of known mutations in
COLE genes in a cohort of NIH patients and found that this COL6A1
intronic mutation was the most common individual mutation. Thus,
this mutation is a newly-discovered single nucleotide polymorphism
(SNP), which results in a mutant form of the alpha 1(VI) chain, and
the presence of this SNP is predictive of an individual developing
a COL6-RD.
[0050] A diagnostic method of this disclosure may generally be
accomplished by obtaining a biological sample from an individual
and analyzing the sample to identify the allele(s) of this
polymorphism carried by the individual. Nucleic acid molecules in
the sample are analyzed to determine the nucleotide present at the
position corresponding to position 189 of intron 11 (SEQ ID NO:3)
[NM_001848 c.930+189) of the gene encoding the alpha 1 chain of
Type VI collagen. The presence of cytosine (C) at the position
corresponding to position 189 of intron 11 indicates the individual
produces normally spliced COL6A1 mRNA. In contrast, the presence of
a thymidine (T) at the position corresponding to position 189 of
intron 11 indicates aberrant splicing COL6A1 pre-mRNA.
[0051] It should be understood that the invention is not limited to
the specific embodiments described herein, as such may vary.
Additionally, the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting on the finally claimed invention, since the scope of the
invention will be limited only by the claims.
[0052] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the
context clearly dictates otherwise. For example, a nucleic acid
molecule refers to one or more nucleic acid molecules. As such, the
terms "a", "an", "one or more" and "at least one" can be used
interchangeably. Similarly, the terms "comprising", "including" and
"having" can be used interchangeably. It is further noted that the
claims may be drafted to exclude any optional element. As such,
this statement is intended to serve as antecedent basis for use of
such exclusive terminology as "solely," "only" and the like, in
connection with the recitation of claim elements, or use of a
"negative" limitation.
[0053] As used herein, a biological sample refers to any fluid or
tissue from an individual that can be analyzed for the presence of
a polymorphism. Preferably, such samples comprise nucleic acid
molecules, such as DNA, cDNA, and/or RNA, including mRNA and miRNA.
Examples of the type of sample that can be used to practice the
methods of this disclosure include, but are not limited to, a blood
sample, a tissue sample (esp. a muscle biopsy), and a buccal swab.
Methods of obtaining such samples are known to those skilled in the
art.
[0054] Once a sample has been obtained, it is analyzed for the
presence or absence of specific alleles (i.e., C or T) of intron 11
from the COL6A1 gene. This may include one or more of detecting the
point mutation in genomic DNA, detecting the presence of the pseudo
exon in RNA transcripts, and/or detecting the 9-amino acid sequence
in the COL6A1 protein produced as a result of the insertion of the
pseudo exon. The presence of the `T` allele (as evidenced by any
one or more of these biomarkers of this SNP) indicates the
individual is at greatly increased risk of developing a
neuromuscular dystrophy, especially Ullrich muscular dystrophy.
[0055] As used herein, the COL6A1 gene, refers to a nucleic acid
sequence encoding an alpha (a) chain of Type VI collagen from a
mammal. One example of a COL6A1 coding sequence is the gene at
position number hg38 chr21:45,981,737-46,005,050 of the human
genome assembly found at genome.ucsc.edu. Another example is the
nucleic acid sequence represented as GenBank Accession No.
BC052575.1. Further, a COL6A1 coding sequence can refer to a
nucleic acid sequence encoding at least a portion of an alpha 1
chain from Type VI collagen. Such a portion can encode a fragment
of the protein (e.g., a 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100
contiguous amino acid segments from any part of the whole protein),
may encode a domain (e.g., a transmembrane domain), may be an exon,
or may refer to the entire protein, including any splicing
variants. COL6A1 genes or coding sequences of this disclosure can
be from any mammal having such a gene or coding sequence. Such
mammals include, but are not limited to, humans, mice, canines,
felines, and equines.
[0056] As used herein, an allele refers to one specific form of a
polymorphism. If a specific sequence contains a polymorphism having
several sequence variations, each unique variation is referred to
as an allele. For example, if a particular position in a nucleotide
sequence in a chromosome contains a cytosine, and the corresponding
position in the homologous chromosome contains a thymidine, such
polymorphism is said to have two alleles. If a third form of the
chromosome exists in which the corresponding position is a guanine,
the polymorphism would be said to have three alleles. Moreover, as
an example of how such alleles can be identified, or differentiated
from one another, the exemplified alleles can be referred to as "a
C allele, a T allele and G allele," respectively. The specific
nucleotide changes at these variant sites that differ between
different alleles are termed variants, mutations, or
polymorphisms.
[0057] One allelic form of a polymorphism may be arbitrarily
designated as the reference form and other allelic forms are
designated as alternative or variant alleles. For example, if a
particular allele is associated with a particular phenotypic
characteristic (e.g., the presence of a disease, the ability to
respond to a disease, the ability to respond to treatment for a
disease, etc.), the beneficial allelic form may be referred to as a
"wild-type form" or beneficial form, while the unfavorable,
disease-associated allelic form can be referred to as the
disadvantageous form, the unfavorable form, the mutant form, the
alternative form, the genetic risk variant, and the like. With
regard to this disclosure, the wild-type (wt) or reference form is
the C allele, and the mutant or genetic risk variant is the T
allele.
[0058] Sequences referred to throughout this application are shown
Table 1 below.
TABLE-US-00001 TABLE 1 SEQ ID NO Sequence Comments 1
GGAGAAAAAGGGAGCCGTGGGGAGAAG Exon 11 2 GEKGSRGEK Amino acid
(GlyGluLysGlySerArgGlyGluLys) sequence of Exon 11 (9 amino acids) 3
gtgagtgaggctcgacctcggagctggtctctccagg Intron 11
cgcagatgtgccatcctggacgagggtgtccccgggg
atgaggacagtgtccctgacaggagaccacgtgtcct
gcagacccgctccaccgcccctcgccgtcccctccat
ctggaaggacaaggacagccacccaggcacccagcaa
aggcgcctgtgtcactttcaccccaccccagagcagg
ggtcccccgggcggttaccctctgcggagccgggggt
cccccgggcggttaccctctgcggagccgggggtccc
ccgggcggttaccctctgcagagcggcccctccccat
cactgtcagtccccatgattctcagcagtgatgttgt
cccctcgggttgggggcacccaagcccctgcctcgcg
tgggcctaagccaggcttgccctgccctccccacccc
aaataccccctcacacccgcttcctgtctccgcag 4
acccgctccaccgcccctcgccgtcccctccatctgg Sequence of
aaggacaaggacagccacccaggcacccagcaaag insertion = pseudo exon (72
base pairs) 5 TRSTAPRRPLHLEGQGQPPRHPAK Amino acid sequence of
pseudo exon (24 aa's) 6 acccaggcacccagcaaagg(c/t)gcctgtgtcact
Mutation site +/-20 ttcacccc bases (C is wildtype; T is mutation)
mutation is at position 21 of SEQ ID NO: 6 7
GGCTCCAGGGGACCCAAGGGCTACAAG Exon 12 8 GSRGPKGYK Amino acid
(GlySerArgGlyProLysGlyTyrLys) sequence of Exon 12 (9 amino acids) 9
CGTGGGGAGAAGacccgctccacc Exon 11/pseudo- exon junction 10 RGEKTRST
Translation of SEQ ID NO: 9 11 cacccagcaaagGGCTCCAGGGGA
Pseudo-exon/exon 12 junction 12 HPAKGSRG Translation of SEQ ID NO:
11 13 CGTGGGGAGAAGGGCTCCAGGGGA Exon 11/exon 12 junction 14 RGEKGSRG
Translation of SEQ ID NO: 13 2'O-Methyl Phosphorothioate
oligonucleotide (2'O-Me) sequences 15 GUGGAGCGGGUCUGCAGGACACGUG
COL6Aps11_A(-14 +11)(ID: 23) 16 GGCUGUCCUUGUCCUCCCAGAUGGA
COL6Aps11_A(+29 +53)(ID: 24) 17 AGGCACCUUUGCUGGGUGCCUGGGU
COL6Aps11_D(+19 -6)(ID: 25) 18 UGAAAGUGACACAGGCACCUUUGCU
COL6Aps11_D(+7 -18)(ID: 26) 19 GGUGAAAGUGACACAGGCAACCU
COL6Aps11_D(+2 -20)(ID: 27) 20 AGAUGGAGGGGACGGCGAGG COL6Aps11_A(+16
+35)(ID: 17) 21 GGCUGUCCUUGUCCUUCCAG COL6Aps11_A(+33 +52)(ID: 18)
22 GUGCCUGGGUGGCUGUCCUU COL6Aps11_A(+37 +56)(ID: 19) Target
Sequences on (alpha 1(Vi) chain) pre-mRNA for SEQ ID Nos 15-22 23
CACGUGUCCUGCAGACCCGCUCCAC Target sequence for COL6Aps11_A(-14 +11)
24 UCCAUCUGGAAGGACAAGGACAGCC Target sequence for COL6Aps11_A(+29
+53) 25 ACCCAGGCACCCAGCAAAGGUGCCU Target sequence for
COL6Aps11_D(+19 -6) 26 AGCAAAGGUGCCUGUGUCACUUUCA Target sequence
for COL6Aps11_D(+7 -18) 27 AGGUGCCUGUGUCACUUUCACC Target sequence
for COL6Aps11_D(+02 -20) 28 CCUCGCCGUCCCCUCCAUCU Target sequence
for COL6Aps11_A(+16 +35) 29 CUGGAAGGACAAGGACAGCC Target sequence
for COL6Aps11_A(+33 +52) 30 AAGGACAGCCACCCAGGCAC Target sequence
for COL6Aps11_A(+37 +56) Phosphorothiamidate morpholino
oligonucleotide (PMO) sequences 31 AGGACACCTGGTCTCCTGTCAGGGA
COL6A1-ex11b-1 (ID:1) 32 GCTGTCCTTGTCCTTCCAGATGGAG COL6A1-ex11b-2
(ID:2) 33 TGAAAGTGACACAGGCACCTTTGCT COL6A1-ex11b-3 (ID:3) 34
GTGGCTGTCCTTGTCCTTCCAGATG COL6A1-ex11b-2b (ID:2b) 35
TTGTCCTTCCAGATGGAcGGGAC COL6A1-ex11b-4 (ID:4) 36
GTGCCTGGGTcGCTGTCCTTGTCCT COL6A1-ex11b-5 (ID:5) New oligo sequences
added to PCT 37 TCTGCAGGACACGTGGTCTCCTGTC COL6A1-intron11- 1b 38
GGTCTGCAGGACACGTGGTCTCCTG COL6A1-intron11- 1c 39
CTGTCCTTGTCCTTCCAGATGGAGG COL6A1-intron11- 2c 40
GGCTGTCCTTGTCCTTCCAGATGGA COL6A1-intron11- 2d 41
TGGCTGTCCTTGTCCTTCCAGATGG COL6A1-intron11- 2e 42
GGTGGCTGTCCTTGTCCTTCCAGAT COL6A1-intron11- 2f 43
TGGGGTGAAAGTGACACAGGCACCT COL6A1-intron11- 3b 44
GGTGAAAGTGACACAGGCACCTTTG COL6A1-intron11- 3c Target Sequences on
(alpha 1(Vi) chain) pre-mRNA for SEQ ID Nos 15-22 45
UCCCUGACAGGAGACCACGUGUCCU Target sequence for COL6A1-ex11b-1 46
CUCCAUCUGGAAGGACAAGGACAGC Target sequence for COL6A1-ex11b-2 47
AGCAAAGGUGCCUGUGUCACUUUCA Target sequence for COL6A1-ex11b-3 48
CAUCUGGAAGGACAAGGACAGCCAC Target sequence for COL6A1-ex11b-2b 49
GUCCCCUCCAUCUGGAAGGACAA Target sequence for COL6A1-ex11b-4 50
AGGACAAGGACAGCCACCCAGGCAC Target sequence for COL6A1-ex11b-5 New
target sequences in PCT 51 gacaggagaccacguguccugcagA Target
sequence for COL6A1-intron11- 1b 52 caggagaccacguguccugcagACC
Target sequence for COL6A1-intron11- 1c 53
CCUCCAUCUGGAAGGACAAGGACAG Target sequence for COL6A1-intron11- 2c
54 UCCAUCUGGAAGGACAAGGACAGCC Target sequence for COL6A1-intron11-
2d 55 CCAUCUGGAAGGACAAGGACAGCCA Target sequence for
COL6A1-intron11- 2e 56 AUCUGGAAGGACAAGGACAGCCACC Target sequence
for COL6A1-intron11- 2f 57 AGgugccugugucacuuucacccca Target
sequence for COL6A1-intron11- 3b 58 CAAAGgugccugugucacuuucacc
Target sequence for COL6A1-intron11- 3c Sequences for alpha 1 (VI)
ORF and protein 59 AGCCGTGGGGAGAAGgtgagtgaggctcga Exon 11/intron 11
junction 60 ttcctgtctccgcagGGCTCCAGGGGACCC Intron 11/exon 12
junction 61 Nucleic acid sequence encoding wt alpha 1 NM_001848
(VI) chain protein 62 MRAARALLPLLLQACWTAAQDEPETPRAVAFQDCPVD
Sequence of wt LFFVLDTSESVALRLKPYGALVDKVKSFTKRFIDNLR alpha 1(VI)
chain DRYYRCDRNLVWNAGALHYSDEVEIIQGLTRMPGGRD protein. Translation
ALKSSVDAVKYFGKGTYTDCAIKKGLEQLLVGGSHLK of exon 11/12
ENKYLIVVTDGHPLEGYKEPCGGLEDAVNEAKHLGVK sequences is
VFSVAITPDHLEPRLSIIATDHTYRRNFTAADWGQSR underlined
DAEEAISQTIDTIVDMIKNNVEQVCCSFECQPARGPP
GLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVG
YQGMKGEKGSRGEKGSRGPKGYKGEKGKRGIDGCDGV
KGEMGYPGLPGCKGSPGFDGIQGPPGPKGDPGAFGLK
GEKGEPGADGEAGRPGSSGPSGDEGQPGEPGPPGEKG
EAGDEGNPGPDGAPGERGGPGERGPRGTPGTRGPRGD
PGEAGPQGDQGREGPVGVPGDPGEAGPIGPKGYRGDE
GPPGSEGARGAPGPAGPPGDPGLMGERGEDGPAGNGT
EGFPGFPGYPGNRGAPGINGTKGYPGLKGDEGEAGDP
GDDNNDIAPRGVKGAKGYRGPEGPQGPPGHQGPPGPD
ECEILDIIMKMCSCCECKCGPIDLLFVLDSSESIGLQ
NFEIAKDFVVKVIDRLSRDELVKFEPGQSYAGVVQYS
HSQMQEHVSLRSPSIRNVQELKEAIKSLQWMAGGTFT
GEALQYTRDQLLPPSPNNRIALVITDGRSDTQRDTTP
LNVLCSPGIQVVSVGIKDVFDFIPGSDQLNVISCQGL
APSQGRPGLSLVKENYAELLEDAFLKNVTAQICIDKK
CPDYTCPITFSSPADITILLDGSASVGSHNFDTTKRF
AKRLAERFLTAGRTDPAHDVRVAVVQYSGTGQQRPER
ASLQFLQNYTALASAVLAMDFINDATDVNDALGYVTR
FYREASSGAAKKRLLLFSDGNSQGATPAAIEKAVQEA
QRAGIEIFVVVVGRQVNEPHIRVLVTGKTAEYDVAYG ESHLFRVPSYQALLRGVFHQTVSRKVALG
63 MRAARALLPLLLQACWTAAQDEPETPRAVAFQDCPVD Sequence of alpha
LFFVLDTSESVALRLKPYGALVDKVKSFTKRFIDNLR 1(VI) chain protein
DRYYRCDRNLVWNAGALHYSDEVEIIQGLTRMPGGRD containing pseudo-
ALKSSVDAVKYFGKGTYTDCAIKKGLEQLLVGGSHLK exon;
ENKYLIVVTDGHPLEGYKEPCGGLEDAVNEAKHLGVK (TRSTAPRRPLHL
VFSVAITPDHLEPRLSIIATDHTYRRNFTAADWGQSR EGQGQPPRHPAK)
DAEEAISQTIDTIVDMIKNNVEQVCCSFECQPARGPP Translations of exon
GLRGDPGFEGERGKPGLPGEKGEAGDPGRPGDLGPVG 11 and exon 12
YQGMKGEKGSRGEKTRSTAPRRPLHLEGQGQPPRHPA sequences are
KGSRGPKGYKGEKGKRGIDGVDGVKGEMGYPGLPGCK underlined.
GSPGFDGIQGPPGPKGDPGAFGLKGEKGEPGADGEAG Translation of
RPGSSGPSGDEGQPGEPGPPGEKGEAGDEGNPGPDGA pseudoexon
PGERGGPGERGPRGTPGTRGPRGDPGEAGPQGDQGRE sequence is bolded
GPVGVPGDPGEAGPIGPKGYRGDEGPPGSEGARGAPG
PAGPPGDPGLMGERGEDGPAGNGTEGFPGFPGYPGNR
GAPGINGTKGYPGLKGDEGEAGDPGDDNNDIAPRGVK
GAKGYRGPEGPQGPPGHQGPPGPDECEILDIIMKMCS
CCECKCGPIDLLFVLDSSESIGLQNFEIAKDFVVKVI
DRLSRDELVKFEPGQSYAGVVQYSHSQMQEHVSLRSP
SIRNVQELKEAIKSLQWMAGGTFTGEALQYTRDQLLP
PSPNNRIALVITDGRSDTQRDTTPLNVLCSPGIQVVS
VGIKDVFDFIPGSDQLNVISCQGLAPSQGRPGLSLVK
ENYAELLEDAFLKNVTAQICIDKKCPDYTCPITFSSP
ADITILLGDSASVGSHNFDTTKRFAKRLAERFLTAGR
TDPAHDVRVAVVQYSGTGQQRPERASLQFLQNYTALA
SAVDAMDFINDATDVNDALGYVTRFYREASSGAAKKR
LLLFSDGNSQGATPAAIEKAVQEAQRAGIEIFVVVVG
RQVNEPHIRVLVTGKTAEYDVAYGESHLFRVPSYQAL LRGVFHQTVSRKVALG
[0059] It is well known that chromosomes are composed of
double-stranded DNA molecules. Thus, while this disclosure refers
to detecting the presence of particular nucleotides in a particular
nucleic acid strand or sequence (e.g., SEQ ID NO:3, SEQ ID NO:4,
SEQ ID NO:6, etc.), this disclosure may also be practiced by
detecting the corresponding nucleotide in a complementary strand.
The newly discovered polymorphism of this disclosure is located in
an intronic segment of the genome (intron 11 of the COL6A1 gene)
which is represented by SEQ ID NO:3.
[0060] Thus, this disclosure provides methods of diagnosing the
likelihood of an individual to develop a Collagen VI related
disorder (COL6-RD). These methods include obtaining a biological
sample from the individual, and analyzing the sample to determine
if the C or T allele is present at a locus in chromosome 21
represented by SEQ ID NO:3. The presence of the T allele indicates
the individual will develop a COL6-RD.
[0061] Another method of this disclosure is a method of diagnosing
the likelihood of an individual to develop a neuromuscular
disorder. This method includes obtaining a biological sample from
the individual, and analyzing the sample to determine if the C or T
allele is present at a locus in chromosome 21 represented by SEQ ID
NO:3. The presence of the T allele indicates the individual will
develop a neuromuscular disorder.
[0062] A related method of this disclosure is a method of
diagnosing the likelihood of an individual to develop Ullrich
muscular dystrophy. This method includes obtaining a biological
sample from the individual, and analyzing the sample to determine
if the C or T allele is present at a locus in chromosome 21
represented by SEQ ID NO:3. The presence of the T allele indicates
the individual will develop Ullrich muscular dystrophy.
[0063] A related method is a method of diagnosing a COL6-RD in an
individual including obtaining a plasma sample from an individual,
separating the sample into a cellular and non-cellular fraction,
detecting the presence of the T allele in the cellular fraction,
and providing a diagnosis of a COL6-RD based on the presence of the
T allele in the cellular fraction of the sample.
[0064] In these methods, as noted above, the biological sample may
include, for example, a blood sample, a tissue sample (esp. a
muscle biopsy), and a buccal swab, and the presence or absence of
the C or T allele may be detected by analyzing genomic DNA, RNA
transcripts, and/or the COL6A1 protein.
[0065] Similarly, this disclosure provides methods of detecting a
SNP in intron 11 of the COL6A1 gene, including obtaining a nucleic
acid sample from an individual that includes a locus in chromosome
21 represented by SEQ ID NO:3, and detecting the presence of the T
allele at a position corresponding to position 21 of SEQ ID
NO:6.
[0066] A related method of detecting a SNP in intron 11 of the
COL6A1 gene, provided by this disclosure, includes obtaining a
plasma sample from a human patient, and detecting whether the T
allele or the C allele is present at a position corresponding to
position 21 of the genomic DNA sequence of SEQ ID NO:6.
[0067] Thus, a related method provided by this disclosure is a
method of detecting Ullrich muscular dystrophy in a patient by
obtaining a plasma sample from a human patient, and detecting
whether the T allele or the C allele is present at a position
corresponding to position 21 of the genomic DNA sequence of SEQ ID
NO:6.
[0068] A number of methods are available for analyzing the presence
or absence of the SNP described in this disclosure, which can be
applied to the COL6A1 region of the genome in a nucleic acid sample
isolated from a biological sample obtained from a subject. Assays
for detection of polymorphisms or mutations fall into several
categories, including but not limited to, direct sequencing assays,
fragment polymorphism assays, hybridization assays, and computer
based data analysis. Protocols and commercially available kits or
services for performing these general methods are available. These
assays may be performed in combination or in hybrid (i.e.,
different reagents or technologies from several assays are combined
to yield one assay). The following assays are useful, and are
described in relationship to detection of the SNP found in the
COL6A1 region of the genome.
[0069] The presence or absence of alleles may be determined using a
direct sequencing technique. In these assays, DNA samples are first
isolated from a subject using any suitable method. DNA in the
region of interest may be amplified using the Polymerase Chain
Reaction (PCR). Alternatively, or additionally, RNA may be used to
generate cDNA and then perform detection analysis of the
polymorphism. Following amplification, DNA or cDNA in the region of
interest (i.e., the region containing the polymorphism) is
sequenced using any suitable method, including but not limited to,
manual sequencing (e.g., using labeled marker nucleotides), or
automated sequencing. The results of the sequencing are displayed
using any suitable method. The sequence is examined and the
presence or absence of a given allele is determined.
[0070] Alleles may also be determined using a PCR-based assay. The
PCR assay comprises the use of oligonucleotide primers to amplify a
fragment containing the polymorphism of interest. Amplification of
a target polynucleotide sequence may be carried out by any method
known to the skilled artisan. Amplification methods include, but
are not limited to, PCR, including real time PCR (RT-PCR), strand
displacement amplification, pyrosequencing, strand displacement
amplification using Phi29 DNA polymerase (U.S. Pat. No. 5,001,050),
transcription-based amplification, self-sustained sequence
replication ("3SR"), the Qbeta replicase system, nucleic acid
sequence-based amplification ("NASBA"), the repair chain reaction
("RCR"), boomerang DNA amplification (or "BDA"), and mismatch PCR.
PCR is the preferred method of amplifying the target polynucleotide
sequence.
[0071] PCR may be carried out in accordance with techniques known
by the skilled artisan. In general, PCR includes first treating a
nucleic acid sample (e.g., in the presence of a heat stable DNA
polymerase) with a pair of amplification primers. One primer of the
pair hybridizes to one strand of a target polynucleotide sequence.
The second primer of the pair hybridizes to the other,
complementary strand of the target polynucleotide sequence. The
primers are hybridized to their target polynucleotide sequence
strands under conditions such that an extension product of each
primer is synthesized which is complementary to each nucleic acid
strand. The extension product synthesized from each primer, when it
is separated from its complement, can serve as a template for
synthesis of the extension product of the other primer. After
primer extension, the sample is treated with denaturing conditions
to separate the primer extension products from their templates.
These steps are cyclically repeated until the desired degree of
amplification is obtained. The amplified target polynucleotide can
then be used in one of the detection assays described herein to
identify the presence or absence of polymorphism of this
disclosure.
[0072] Because mismatches between the primer sequence and the
template sequence can result in inability of the polymerase to
extend the primer, and thus failure to generate an amplification
product, primers designed to hybridize perfectly with one or more
allele can be used to detect such alleles. While mismatches can be
designed at any position on the primer, mismatches at the 3'
terminal end of the primer are most beneficial as such primers
usually cannot be extended by the polymerase. For example, a primer
consisting of 21 nucleotides, the first 20 of which are identical
to nucleotides 1-20 of SEQ ID NO:6, the 21.sup.st nucleotide being
a cytosine, would successfully produce a PCR amplification product
from template DNA comprising SEQ ID NO:3. Alternatively, a primer
consisting of 21 nucleotides, the first 20 of which are identical
to nucleotides 1-20 of SEQ ID NO:6, the 21.sup.st nucleotide being
a thymidine, would only produce a PCR amplification product from
template DNA comprising SEQ ID NO:3 if the SNP of this disclosure
were present. Thus, use of such primers would discriminate between
DNA comprising wildtype COL6A1 and COL6A1 in which the T allele is
present.
[0073] The SNP may also be detected using a fragment length
polymorphism assay, in which a unique DNA banding pattern based on
cleaving the DNA at a series of positions is generated using an
enzyme (e.g., a restriction endonuclease). DNA fragments from a
sample containing a polymorphism will have a different banding
pattern than wild type.
[0074] The SNP may also be detected by fragment sizing analysis.
Such analysis can be performed using, for example, the Beckman
Coulter CEQ 8000 genetic analysis system, a method well-known in
the art for microsatellite polymorphism determination.
[0075] The SNP may also be detected using a restriction fragment
length polymorphism assay (RPLP). The region of interest is first
isolated using PCR. The PCR products are then cleaved with
restriction enzymes known to give a unique length fragment for a
given polymorphism. The restriction-enzyme digested PCR products
are separated by agarose gel electrophoresis and visualized by
ethidium bromide staining, or other means know in the art, and
compared to controls (wild-type).
[0076] The SNP may also be detected using a CLEAVASE fragment
length polymorphism assay (CFLP; Third Wave Technologies, Madison,
Wis.: see e.g., U.S. Pat. No. 5,888,750). This assay is based on
the observation that, when single strands of DNA fold on
themselves, they assume higher order structures that are highly
individual to the precise sequence of the DNA molecule. These
secondary structures involve partially duplexed regions of DNA such
that single stranded regions are juxtaposed with double stranded
DNA hairpins. The CLEAVASE I enzyme is a structure-specific,
thermostable nuclease that recognizes and cleaves the junctions
between these single-stranded and double-stranded regions. Such
assay is exemplified in Oldenburg, M. C., Siebert, M., "New
Cleavase Fragment Length Polymorphism Method Improves the Mutation
Detection Assay" 2000 Biotechniques 28:351-357.
[0077] The SNP may also be detected by hybridization assay, in
which the presence or absence of a given allele or mutation is
determined based on the ability of the DNA from the sample to
hybridize to a complementary DNA molecule (e.g., an oligonucleotide
probe). A variety of hybridization assays using a variety of
technologies for hybridization and detection are available. The
hybridized nucleic acids may be detected using one or more labels
attached to the sample nucleic acids. The labels may be
incorporated by any of a number of means well known to those of
skill in the art. In one embodiment, the label is simultaneously
incorporated during the amplification step in the preparation of
the sample nucleic acids. For example, PCR can be performed using
labeled primers or labeled nucleotides, resulting in a labeled
amplification product. Additionally, or alternatively,
transcription amplification using a labeled nucleotide (e.g.
fluorescein-labeled UTP and/or CTP) incorporates a label into the
transcribed nucleic acids. Alternatively, a label may be added
directly to the original nucleic acid sample (e.g., mRNA, polyA
mRNA, cDNA, genomic DNA etc.) or to the amplification product after
the amplification is completed. Means of attaching labels to
nucleic acids are well known to those of skill in the art and
include, for example, nick translation or end-labeling (e.g. with a
labeled RNA) by kinasing the nucleic acid and subsequent attachment
(ligation) of a nucleic acid linker joining the sample nucleic acid
to a label (e.g., a fluorophore). A label may also be added to the
end of fragments using terminal deoxytransferase (TdT).
[0078] Detectable labels suitable for use in the methods of this
disclosure include any composition detectable by spectroscopic,
photochemical, biochemical, immunochemical, electrical, optical or
chemical means. Useful labels include, but are not limited to:
biotin for staining with labeled streptavidin conjugate;
anti-biotin antibodies; magnetic beads (e.g., Dynabeads.TM.);
fluorescent, dyes (e.g., fluorescein, Texas Red, rhodamine, green
fluorescent protein, and the like); radiolabels (e.g., H.sup.3,
I.sup.125, S.sup.35, C.sup.14, or P.sup.32); phosphorescent labels;
enzymes (e.g., horse radish peroxidase, alkaline phosphatase and
others commonly used in an ELISA); and calorimetric labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.) beads. Other labels are known to those
skilled in the art.
[0079] Means of detecting such labels are well known to those of
skill in the art. Thus, for example, radiolabels may be detected
using photographic film or scintillation counters; fluorescent
markers may be detected using a photodetector to detect emitted
light. Enzymatic labels are typically detected by providing the
enzyme with a substrate and detecting the reaction product produced
by the action of the enzyme on the substrate, and calorimetric
labels are detected by simply visualizing the colored label.
[0080] The label may be added to the target nucleic acid(s) prior
to, or after the hybridization. So-called "direct labels" are
detectable labels that are directly attached to or incorporated
into the target nucleic acid prior to hybridization. In contrast,
so-called "indirect labels" are joined to the hybrid duplex after
hybridization. Often, the indirect label is attached to a binding
moiety that has been attached to the target nucleic acid prior to
the hybridization. Thus, for example, the target nucleic acid may
be biotinylated before the hybridization. After hybridization, an
avidin-conjugated fluorophore will bind the biotin bearing hybrid
duplexes providing a label that is easily detected. For a detailed
review of methods of labeling nucleic acids and detecting labeled
hybridized nucleic acids, see Tijssen, 1993, Laboratory Techniques
in Biochemistry and Molecular Biology, Vol. 24; Hybridization with
Nucleic Acid Probes.
[0081] Hybridization of a probe to the sequence of interest (e.g.,
polymorphism) may be detected directly by visualizing a bound probe
(e.g., a Northern or Southern assay; See e.g., Ausabel et al.
(Eds.), 1991, Current Protocols in Molecular Biology, John Wiley
& Sons, NY). In an example of such assays, genomic DNA
(Southern) or RNA (Northern) is isolated from a subject. The DNA or
RNA is then cleaved with a series of restriction enzymes that
cleave infrequently in the genome and not near any of the markers
being assayed. The DNA or RNA is then separated (e.g., agarose gel
electrophoresis) and transferred to a membrane. A labeled (e.g., by
incorporating a radionucleotide) probe or probes specific for the
mutation being detected is allowed to contact the membrane under a
condition of low, medium, or high stringency conditions. Unbound
probe is removed and the presence of binding is detected by
visualizing the labeled probe.
[0082] The SNP may also be detected using a DNA chip hybridization
assay, in which a series of oligonucleotide probes are affixed to a
solid support. The oligonucleotide probes are designed to be unique
to a given single nucleotide polymorphism. The DNA sample of
interest is contacted with the DNA "chip" and hybridization is
detected. An example of such technology is a GeneChip (Affymetrix,
Santa Clara, Calif.; see e.g., U.S. Pat. No. 6,045,996) assay. The
GeneChip technology uses miniaturized, high-density arrays of
oligonucleotide probes affixed to a "chip." Probe arrays are
manufactured by Affymetrix's light-directed chemical synthesis
process, which combines solid-phase chemical synthesis with
photolithographic fabrication techniques employed in the
semiconductor industry. Using a series of photolithographic masks
to define chip exposure sites, followed by specific chemical
synthesis steps, the process constructs high-density arrays of
oligonucleotides, with each probe in a predefined position in the
array. Multiple probe arrays are synthesized simultaneously on a
large glass wafer. The wafers are then diced, and individual probe
arrays are packaged in injection-molded plastic cartridges, which
protect them from the environment and serve as chambers for
hybridization.
[0083] The nucleic acid to be analyzed is isolated from a
biological sample obtained from the subject, amplified by PCR, and
labeled with a fluorescent reporter group. The labeled DNA is then
incubated with the array using a fluidics station. The array is
then inserted into the scanner, where patterns of hybridization are
detected. The hybridization data are collected as light emitted
from the fluorescent reporter groups already incorporated into the
target, which is bound to the probe array. Probes that perfectly
match the target generally produce stronger signals than those that
have mismatches. Because the sequence and positions of each probe
on the array are known, by complementarity, the identity of the
target nucleic acid applied to the probe array can be
determined.
[0084] The SNP may also be detected using a DNA microchip
containing electronically captured probes. One example of such
technology is a NanoChip (Nanogen, San Diego, Calif.; see e.g.,
U.S. Pat. No. 6,068,818). Through the use of microelectronics, this
technology enables the active movement and concentration charged
molecules to and from designated test sites on its semiconductor
microchip. DNA capture probes unique to a given polymorphism or
mutation are electronically placed at, or "addressed" to, specific
sites on the microchip. Since DNA has a strong negative charge, it
can be electronically moved to an area of positive charge. First, a
test site or a row of test sites on the microchip is electronically
activated with a positive charge. Next, a solution containing the
DNA probes is introduced onto the microchip. The negatively charged
probes rapidly move to the positively charged sites, where they
concentrate and are chemically bound to a site on the microchip.
The microchip is then washed and another solution of distinct DNA
probes is added until the array of specifically bound DNA probes is
complete. A test sample is then analyzed for the presence of target
DNA molecules by determining which of the DNA capture probes
hybridize to complementary DNA in the test sample (e.g., a PCR
amplified gene of interest). An electronic charge is also used to
move and concentrate target molecules to one or more test sites on
the microchip. The electronic concentration of sample DNA at each
test site promotes rapid hybridization of sample DNA with
complementary capture probes (hybridization may occur in minutes).
To remove any unbound nonspecifically bound DNA from each site, the
polarity or charge of the site is reversed to negative, thereby
forcing any unbound or nonspecifically bound DNA back into solution
away from the capture probes. A laser-based fluorescence scanner is
used to detect binding.
[0085] The SNP may also be detected using a "bead array" for the
detection of polymorphisms (Illumina, San Diego, Calif.; see e.g.,
PCT Publications WO99/67641 and WO00/39587, which are herein
incorporated by reference). Illumina uses a bead array technology
that combines fiber optic bundles and beads that self-assemble into
an array. Each fiber optic bundle contains thousands to millions of
individual fibers depending on the diameter of the bundle. The
beads are coated with an oligonucleotide specific for the detection
of a given polymorphism or mutation. Batches of beads are combined
to form a pool specific to the array. To perform an assay, the bead
array is contacted with a prepared subject sample (e.g., DNA).
Hybridization is detected using any suitable method, such as for
example, Enzymatic Detection of Hybridization
[0086] Genomic profiles may be generated using an assay that
detects hybridization by enzymatic cleavage of specific structures.
One example of such an assay is the INVADER.RTM. assay (Third Wave
Technologies; see e.g., U.S. Pat. No. 6,001,567, and Olivier, M.,
The Invader assay for SNP Genotyping, 2005 Mutat. Res. June 3;
573(1-2):103-110, both of which are incorporated herein by
reference). The INVADER.TM. assay detects specific DNA and RNA
sequences by using structure-specific enzymes to cleave a complex
formed by the hybridization of overlapping oligonucleotide probes.
Elevated temperature and an excess of one of the probes enable
multiple probes to be cleaved for each target sequence present
without temperature cycling. These cleaved probes then direct
cleavage of a second labeled probe. The secondary probe
oligonucleotide can be 5'-end labeled with fluorescein that is
quenched by an internal dye. Upon cleavage, the dequenched
fluorescein labeled product may be detected using a standard
fluorescence plate reader.
[0087] A MassARRAY system (Sequenom, San Diego, Calif.) may be used
to detect polymorphisms (see e.g., U.S. Pat. No. 6,043,031).
[0088] Genomic DNA samples are usually, but need not be, amplified
before being analyzed. Genomic DNA can be obtained from any
biological sample. Amplification of genomic DNA generates a single
species of nucleic acid if the individual from whom the sample was
obtained is homozygous at the polymorphic site, or two species of
nucleic acid if the individual is heterozygous.
[0089] RNA samples also are often subject to amplification. In this
case, amplification is typically, but not necessarily, proceeded by
reverse transcription. Amplification of all expressed mRNA can be
performed as described in Innis et al., 1990. Academic Press. "PCR
Protocols: A Guide to Methods and Applications"; and Bustin,
Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction assays, Journal of
Molecular Endocrinology 25:169-193, 2000. Amplification of an RNA
sample from a diploid sample can generate two species of target
molecules if the individual providing the sample is heterozygous at
a polymorphic site occurring within the expressed RNA, or possibly
more if the species of the RNA is subjected to alternative
splicing. Amplification generally can be performed using the
polymerase chain reaction (PCR) methods known in the art. Nucleic
acids in a target sample can be labeled in the course of
amplification by inclusion of one or more labeled nucleotides in
the amplification mixture. Labels also can be attached to
amplification products after amplification (e.g., by end-labeling).
The amplification product can be RNA or DNA, depending on the
enzyme and substrates used in the amplification reaction. Once a
sample has been analyzed to determine which allele of a
polymorphism is present, the individual can be selected, or
identified, as having a significantly increased risk of developing
a neuromuscular disorder, particularly Ullrich muscular dystrophy,
if the SNP of this disclosure is present in one or both alleles of
COL6A1.
Therapeutic Compositions
[0090] As previously described, the inventors have discovered a
newly identified mutation in the COL6A1, that such mutated gene
encodes a mutated form of the Type VI collagen alpha 1 chain, and
that individuals possessing such mutation develop neuromuscular
disorders. Based on this discovery, the inventors have developed a
novel method of treatment for the above-described collagen
VI-related disorder, the method involving re-directing splicing of
the mutated COL6A1 pre-mRNA so that it undergoes normal splicing.
Such methods may be accomplished by administering to the individual
a therapeutic compound that binds to the COL6A1 pre-mRNA molecule,
thereby preventing use of the newly introduced 5' splice donor
site, and forcing the cellular splicing apparatus to use the normal
splice donor site (i.e., exon 11 splice donor site) and the normal
splice acceptor site (i.e., the exon 12 splice acceptor site). The
resulting mature mRNA molecule lacks the afore-mentioned
pseudo-exon and thus encodes a normal alpha 1(VI) chain
protein.
[0091] As used herein, pre-mRNA refers to messenger RNA (mRNA)
transcribed from the genome, which has not yet undergone splicing.
As used herein, mature mRNA refers to pre-mRNA that has completed
the splicing process and is ready to undergo translation to produce
the encoded protein.
[0092] With regard to the present invention, normal splicing refers
to joining of the native exon 11 splice donor site to the native
exon 12 splice acceptor site. Following normal splicing of wild
type COL6A1 pre-mRNA, exon 11 is joined, in-frame, directly to exon
12, with no intervening coding sequence. As used herein, a native
splice donor or splice acceptor site is a splice donor, or
acceptor, site that is used most commonly, or exclusively, during
splicing of a pre-mRNA molecule transcribed from a wild-type (wt)
gene. For example, in a pre-mRNA molecule from a wild-type COL6A1
gene, the AGgt at the 3' end of exon 11 is the splice donor site
used to join exon 11 to exon 12. The agGG sequence at the 5' end of
exon 12 serves as the splice acceptor site for exon 12. After
splicing, the splice junction has the sequence AGGG. These sites
are illustrated in FIG. 6 (top diagram labeled "normal"). As used
herein, a non-native splice donor or splice acceptor site is a
splice donor, or acceptor, site that is not present in mRNA
transcribed from a wild-type gene, such as wt COL6A1. Non-native
splice donor or splice acceptor sites arise because of one or more
alterations or mutations in the wild-type gene that result in the
formation of a new splice donor or splice acceptor site. For
example, as illustrated in FIG. 6, a mutation of the nucleotide at
position 189 of intron 11 (SEQ ID NO:3) alters the sequence aggc to
aggt, which creates a new splice donor site. This new site is
considered a non-native splice donor site. As previously described,
the cellular splicing apparatus joins this site with the splice
acceptor site at the beginning of exon 12. Consequently, the splice
donor site at the 3' end of exon 11, which is normally joined to
the exon 12 acceptor site, is joined to an acceptor site 115 bp
downstream of the 3' end of exon 11. This downstream acceptor site
is referred to as a cryptic site, since it is present in the wt
pre-mRNA but is not normally used during splicing.
[0093] With regard to this disclosure, aberrant splicing, or mutant
splicing, refers to joining of a non-native splice donor, or splice
acceptor, site to a native (or non-native) splice donor, or
acceptor, site. For example, with regard to the present invention,
the 189C>T mutation introduced a non-native splice donor site
that is then joined to the exon 12 splice acceptor site. Such
splicing can be referred to as aberrant splicing. In addition,
aberrant splicing also includes joining of the exon 11 splice donor
site to the cryptic splice acceptor lying 115 bp downstream of the
3' end of exon 11. In regard to the present invention, the result
of aberrant splicing is that a pseudo-exon is incorporated into the
mature COL6A1 mRNA (see FIG. 6).
[0094] Antisense technology has been demonstrated to be an
effective method of modifying the expression levels of gene
products (see, for example, U.S. Pat. Nos. 8,765,703, 8,946,183,
and U.S. Patent Publication No. 2015/0376615, incorporated herein
by reference in their entirety). Antisense technology works by
interfering with known steps in the normal processing of mRNA.
Briefly, RNA molecules are transcribed from genomic DNA in the
nucleus of the cell. These newly synthesized mRNA molecules, called
primary mRNA or pre-mRNA, must be processed prior to transport to
the cytoplasm for translation into protein at the ribosome. Such
processing includes the addition of a 5' methylated cap and the
addition of a poly(A) tail to the 3' end of the mRNA.
[0095] Maturation of 90-95% of mammalian mRNAs then occurs with
splicing of the precursor (pre-) mRNA. Introns (or intervening
sequences) are regions of a primary transcript (or the DNA encoding
it) that are not included in the coding sequence of the mature
mRNA. Exons (expressed sequences) are regions of a primary
transcript (or the DNA encoding it) that remain in the mature mRNA
when it reaches the cytoplasm. During the splicing process, exons
in the pre-mRNA molecule are spliced together to form the mature
mRNA sequence. Splice junctions, also referred to as splice sites,
are utilized by cellular apparatus to determine which sequences are
removed and where the ends to be joined start and stop. Sequences
on the 5' side of the junction are called the 5' splice site, or
splice donor site, whereas sequences on the 3' side the junction
are referred to as the 3' splice site, or the splice acceptor site.
In splicing, the 3' end of an upstream exon is joined to the 5' end
of the downstream exon. Thus, the un-spliced RNA (or pre-mRNA) has
an exon/intron junction at the 5' end of an intron and an
intron/exon junction at the 3' end of an intron. After the intron
is removed, the exons are contiguous at what is sometimes referred
to as the exon/exon junction or boundary in the mature mRNA.
Cryptic splice sites are those which are less often used but may be
used when the usual splice site is blocked or unavailable. The use
of different combinations of exons by the cell can result in
multiple mRNA transcripts from a single gene.
[0096] Antisense technology can also be used to affect splicing of
a gene transcript. In this application, the antisense
oligonucleotide binds to a pre-spliced RNA molecule (pre-messenger
RNA or pre-mRNA) and re-directs the cellular splicing apparatus,
thereby resulting in modification of the exon content of the
spliced mRNA molecule. Thus, the overall sequence of a protein
encoded by the modified mRNA differs from a protein translated from
mRNA, the splicing of which was not altered. The protein that is
translated from the altered mRNA may be truncated and/or it may be
missing amino acid sequences. Typically, the compounds used to
affect splicing are, or contain, oligonucleotides having a base
sequence complementary to the mRNA being targeted. Such
oligonucleotides are referred to herein as "antisense
oligonucleotides" (AONs).
[0097] A therapeutic compound of this disclosure is a compound
that, upon administration to an individual possessing the
afore-mentioned mutation, results in normal splicing of COL6A1
pre-mRNA molecules, and production of normal alpha 1(VI) chain
protein. These therapeutic compounds may comprise antisense
oligomers targeted to the COL6A1 pre-mRNA so that the newly
introduced splice donor site (i.e., non-native splice site) in the
COL6A1 pre-mRNA is not used, and the COL6A1 pre-mRNA molecule
undergoes normal splicing.
[0098] One aspect of the invention is an antisense oligomer
targeted to a sequence in an intron of a COL6A1 pre-mRNA molecule,
wherein hybridization of the antisense oligomer to the target
sequence in the COL6A1 pre-mRNA molecule results in normal splicing
of the COL6A1 pre-mRNA molecule. In certain aspects, the COL6A1
pre-mRNA molecule comprises a non-native splice donor or splice
acceptor site in the intron. In certain aspects, the presence of
the non-native splice donor or splice acceptor site causes aberrant
splicing of the COL6A1 pre-mRNA molecule. In certain aspects,
hybridization of the antisense oligomer to the target sequence in
the COL6A1 pre-mRNA molecule prevents use of a non-native splice
donor or splice acceptor site.
[0099] As used herein, an antisense oligomer is a polymeric
molecule comprising nucleobases that is capable of hybridizing to a
sequence in a nucleic acid molecule, such as a pre-mRNA or mRNA
molecule. In this regard, the ability to hybridize represents the
antisense activity of the antisense oligomer. The term nucleobase,
as used herein, refers to the heterocyclic base portion of a
nucleoside. In general, a nucleobase is any group that contains one
or more atoms, or groups of atoms, capable of hydrogen bonding to
one or more atoms in the base of another nucleoside. In addition to
"unmodified" or "natural" nucleobases such as the purine
nucleobases adenine (A) and guanine (G), and the pyrimidine
nucleobases thymine (T), cytosine (C) and uracil (U), modified
nucleobases or nucleobase mimetics known to those skilled in the
art are also amenable to the invention. The term "modified
nucleobase" refers to a nucleobase that is similar in structure to
the parent nucleobase, such as for example, a 7-deaza purine, a
5-methyl cytosine, a G-clamp, or a tricyclic phenoxazine nucleobase
mimetic. Methods for preparation of these modified nucleobases are
well known to those skilled in the art.
[0100] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base (e.g., a nucleobase or simply a "base"). The two
most common classes of such heterocyclic bases are purines and the
pyrimidines. Nucleotides are nucleosides that include a phosphate
group covalently linked to the sugar portion of the nucleoside. For
those nucleosides that include a pentofuranosyl sugar, the
phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety
of the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. Within oligonucleotides, the phosphate
groups are commonly referred to as forming the internucleoside
backbone of the oligonucleotide. The normal linkage or backbone of
RNA and DNA is a 3' to 5' phosphodiester linkage.
[0101] The term oligomer includes oligonucleotides,
oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics
and chimeric combinations thereof. Oligomers of the invention
include, but are not limited to, primers, probes, antisense
compounds, antisense oligonucleotides, antisense RNA, antisense
DNA, external guide sequence (EGS) oligonucleotides, alternate
splicers, and siRNAs. As such, these compounds can be introduced in
the form of single-stranded, double-stranded, circular, branched or
hairpins and can contain structural elements such as internal or
terminal bulges or loops.
[0102] Oligomers may be any length suitable for administering to a
cell or individual in order to modulate splicing of an mRNA
molecule. For example, antisense oligomers of this disclosure may
comprise from about 10 to about 50 nucleobases (i.e. from about 10
to about 50 linked nucleosides). This embodies antisense oligomers
of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, or 50 nucleobases. Antisense oligomers
of the invention may comprise, or consist of, 10 to 30 nucleobases,
or 10 to 25 nucleobases. In one embodiment of the invention,
antisense oligomers of the invention comprise, or consists of, 18
nucleobases, 19 nucleobases, 20 nucleobases, 21 nucleobases, 22
nucleobases, 23 nucleobases, 24 nucleobases, 25 nucleobases, 26
nucleobases, 27 nucleobases, 28 nucleobases, 29 nucleobases or 30
nucleobases Methods of determining the optimal length for antisense
oligomers of the invention are known to those skilled in the
art.
[0103] It is understood in the art that RNA molecules often have a
short half-life, making their use as therapeutic agents
problematic. Thus, it is often preferable to include chemical
modifications in oligonucleotides to alter their activity. Chemical
modifications can alter oligomer activity by, for example,
increasing affinity of an antisense oligomer for its target RNA,
increasing nuclease resistance (e.g., resistance to ribonucleases
such as RNaseH), and/or altering the pharmacokinetics (e.g.
half-life) of the oligomer. Thus, for example, it is possible to
replace sugars, nucleobases and/or internucleoside linkages with a
group that maintains the ability of the oligomer to hybridize to
its target sequence, but which impart one or more desirable
characteristics, such as resistance to degradation or increased
half-life, to the oligomer. Such groups can be referred to as
analogs (e.g., sugar analog, nucleobase analog, etc.). Generally,
an analog is used in place of the sugar or sugar-internucleoside
linkage combination, and the nucleobase is maintained for
hybridization to a selected target. Representative examples of a
sugar mimetic include, but are not limited to, cyclohexenyl or
morpholino. Representative examples of a mimetic for a
sugar-internucleoside linkage combination include, but are not
limited to, peptide nucleic acids (PNA) and morpholino groups
linked by uncharged, achiral linkages. In some instances, an analog
is used in place of the nucleobase. Representative nucleobase
mimetics are well known in the art and include, but are not limited
to, tricyclic phenoxazine analogs and universal bases (Berger et
al., Nuc. Acid Res. 2000, 28:2911-14, incorporated herein by
reference). Examples of such sugar, nucleoside and nucleobase
mimetics are disclosed in U.S. Pat. Nos. 8,765,703 and 8,946,183,
both of which are incorporated herein by reference in their
entirety). Methods of synthesis of sugar, nucleoside and nucleobase
mimetics, and the use of such mimetics to produce oligonucleotides
are well known to those skilled in the art.
[0104] Oligomers of the invention can also be conjugated to
cell-penetrating peptides (CPPs). Such peptides are short peptides
that enhance the cellular uptake of oligomers to which they are
attached. CPPs and their use to enhance cellular uptake are known
to those skilled in the art, and are also described in U.S. Pat.
No. 9,303,076, which is incorporated herein by reference.
[0105] As used herein, the terms targeted to, targeting, and the
like, refer to a process of designing an antisense oligomer so that
it specifically hybridizes with a desired nucleic acid molecule,
such as a desired pre-mRNA or mRNA molecule. The terms
"hybridizes," "hybridization," "hybridize to," and the like, are
terms of art that refer to the pairing of nucleobases in
complementary strands of oligonucleotides (e.g., an antisense
oligomer and a selected/target sequence in a pre-mRNA molecule).
While embodiments of this disclosure are not limited to a
particular pairing mechanism, the most common mechanism of pairing
involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or
reversed Hoogsteen hydrogen bonding, between complementary
nucleoside or nucleotide bases (nucleobases). For example, the
natural base adenine is complementary to the natural nucleobases
thymidine and uracil, which pair through the formation of hydrogen
bonds. Similarly, the natural base guanine is complementary to the
natural bases cytosine and 5-methyl cytosine.
[0106] In the context of the invention, the phrase "specifically
hybridizes" refers to the capacity of an antisense oligomer of the
invention to preferentially binds COL6A1 mRNA molecule (e.g.,
pre-mRNA) rather than binding a mRNA molecule encoding a protein
unrelated in structure to the alpha 1 chain of Type VI collagen.
Further, an antisense oligomer that preferentially binds a COL6A1
mRNA molecule is one that hybridizes with a sequence in an mRNA
encoding alpha 1(VI) chain protein (e.g., an alpha 1(VI) chain
pre-mRNA), but which does not significantly hybridize with an mRNA
molecule encoding a protein unrelated in structure to an alpha
1(VI) chain protein. In this context, significant hybridization
refers to binding of an antisense oligomer of the invention with an
affinity or avidity sufficiently high enough to interfere with the
ability of the antisense oligomer to achieve the desired effect.
Examples of such desired effects include, but are not limited to,
modulation of splicing of a COL6A1 pre-mRNA, reduced production of
an aberrant alpha 1(VI) chain, increased production in normal alpha
1(VI) chain or a reduction in symptoms of a COL6A1-related
disorder. Thus, it will be understood by those skilled in the art
that an antisense oligomer is considered specific for a COL6A1 mRNA
molecule (i.e., specifically hybridizes with) when there is a
sufficient degree of complementarity between the linear sequence of
nucleobases in the antisense oligomer and a linear sequence of
nucleobases (target sequence) in the mRNA molecule, to avoid
significant binding of the antisense oligomer to non-target
sequences under conditions in which specific binding is desired
(i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays).
[0107] A used herein, the terms complement, complementary,
complementarity, and the like, refer to the capacity for precise
pairing between nucleobases in an antisense oligomer and
nucleobases in a target sequence. Thus, if a nucleobase (e.g.,
adenine) at a specific position in an antisense oligomer is capable
of hydrogen bonding with a nucleobase (e.g., uracil) at a specific
position in a target sequence, then the nucleobases at those
specific positions in the antisense oligomer and the target
sequence are considered complementary. Usually, the terms
complement, complementary, complementarity, and the like, are
viewed in the context of a comparison between a defined number of
contiguous nucleotides in a first nucleic acid molecule (e.g., an
oligomer) and a similar number of contiguous nucleotides in a
second nucleic acid molecule (e.g., a mRNA molecule), rather than
in a single base to base manner. For example, if an antisense
oligomer is 25 nucleotides in length, its complementarity with a
target sequence is usually determined by comparing the sequence of
the entire oligomer, or a defined portion thereof, with a number of
contiguous nucleotides in a target sequence. An oligomer and a
target sequence are complementary to each other when a sufficient
number of corresponding positions in each molecule are
complementary. Positions in two separate nucleic acid molecules are
considered corresponding if, when the sequences of the two separate
nucleic acid molecules are aligned, the nucleobases at those
positions are adjacent to one another. As an example, when
comparing the sequence of an oligomer to a similarly sized sequence
in a target sequence, the first nucleotide in the oligomer is
compared with a chosen nucleotide at the start of the target
sequence. The second nucleotide in the oligomer (3' to the first
nucleotide) is then compared with the nucleotide directly 3' to the
chosen start nucleotide. This process is then continued with each
nucleotide along the length of the oligomer. Thus, the terms
"specifically hybridizable" and "complementary" are terms which are
used to indicate a sufficient degree of precise pairing or
complementarity over a sufficient number of contiguous nucleobases
such that stable and specific binding occurs between the antisense
compound and a target nucleic acid.
[0108] Hybridization conditions under which a first nucleic acid
molecule will specifically hybridize with a second nucleic acid
molecule are commonly referred to in the art as stringent
hybridization conditions. It is understood by those skilled in the
art that stringent hybridization conditions are sequence-dependent
and can be different in different circumstances. Thus, stringent
conditions under which an oligomer of the invention specifically
hybridizes to a target sequence are determined by the
complementarity of the oligomer sequence and the target sequence
and the nature of the assays in which they are being investigated.
Persons skilled in the relevant art are capable of designing
complementary sequences that specifically hybridize to a particular
target sequence for a given assay or a given use.
[0109] The process of designing an antisense oligomer that is
targeted to a nucleic acid molecule usually begins with
identification of a target nucleic acid molecule, the expression or
splicing of which is to be modulated, and determining the sequence
of the target nucleic acid molecule. In the present invention, the
target nucleic acid molecule is a COL6A1 pre-mRNA molecule.
[0110] The next step in the process of designing an antisense
oligomer targeted to COL6A1 pre-mRNA molecule is the identification
of a target sequence in the mRNA molecule. As used herein, a target
sequence is a nucleic acid sequence in a COL6A1 pre-mRNA molecule
to which an antisense oligomer of the invention will specifically
hybridize, wherein such binding results in normal splicing of the
COL6A1 pre-mRNA molecule. Any sequence in the COL6A1 pre-mRNA
molecule can serve as a target sequence, as long as binding of such
sequence by the antisense oligomer modulates splicing of the COL6A1
pre-mRNA molecule. Preferably, binding of a target sequence by the
antisense oligomer results in normal splicing of the COL6A1
pre-mRNA molecule. Preferred sequences to target are those that
result in normal splicing of exon 11 to exon 12. In this regard,
the inventors have demonstrated that sequences between exon 11 and
exon 12 (i.e., intron 11) in the pre-mRNA molecule can be used as
target sequences to effect normal splicing of COL6A1 pre-mRNA
molecules.
[0111] Once a target sequence has been identified, the antisense
oligomer is designed to include a nucleobase sequence sufficiently
complementary to the target sequence so that the antisense oligomer
specifically hybridizes to the target sequence in the COL6A1
pre-mRNA molecule. It is well known in the art that the greater the
degree of complementarity between two nucleic acid sequences, the
stronger and more specific is the hybridization interaction. It is
also well understood that the strongest and most specific
hybridization occurs between two nucleic acid molecules that are
fully complementary. As used herein, the term fully complementary
refers to a situation when each nucleobase in a nucleic acid
sequence is capable of hydrogen binding with the nucleobase in the
corresponding position in a second nucleic acid molecule. For
example, a nucleic acid molecule having the sequence 5'-ACUGA-3' is
fully (100%) complementary to a nucleic acid molecule having the
sequence 3'-UGACU-5'. Likewise, a nucleic acid molecule having the
sequence 5'-ACUGACACGU-3' is 90% complementary to a nucleic acid
molecule having the sequence 3'-UAACUGUGCA-5', and 80%
complementary to a nucleic acid molecule having the sequence
3'-UAACUUUGCA-5'. Such examples demonstrate the concept of percent
complementarity.
[0112] Thus, the targeting sequence may be fully complementary to
the target sequence. The antisense oligomer may comprise an at
least 6 contiguous nucleobase region that is fully complementary to
an at least 6 contiguous nucleobase region in the target sequence.
Similarly, the antisense oligomer may comprise an at least 8, 10,
12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, or 27-contiguous
nucleobase sequence that is fully complementary to an at least 8,
10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, or 27-contiguous
nucleobase sequence in the respective target sequence.
[0113] Each nucleobase in the antisense oligomer may be
complementary to the nucleobase at the corresponding position in
the target sequence, or only some nucleobases at corresponding
positions may be complementary. For example, in an antisense
oligomer consisting of 30 nucleotides, all 30 nucleotides can be
complementary to a 30 contiguous nucleotide target sequence.
Alternatively, a 30-mer antisense oligomer may comprise only 20
contiguous nucleotides that are complementary to 20-contiguous
nucleotides in the target sequence, with the remaining 10
nucleotides in the oligomer being mismatched to nucleotides outside
of the target sequence. Oligomers of the invention may have a
targeting sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, or 27-contiguous nucleobases that
are fully complementary to the same number of contiguous
nucleobases in the target sequence.
[0114] The inclusion of mismatches between nucleobases of an
antisense oligomer and nucleobases of a target sequence is possible
without eliminating the activity of the oligomer (e.g., modulation
of splicing). Moreover, such mismatches can occur at any location
in the interaction between the antisense oligomer and the target
sequence, so long as the antisense oligomer is capable of
specifically hybridizing to the targeted nucleic acid molecule.
Thus, antisense oligomers of the invention may comprise up to about
20% of nucleotides that are mismatched, as long as the antisense
oligomer specifically hybridizes to the target sequence. Thus,
antisense oligomers comprise no more than 20%, 15%, 10%, 5% or 3%
mismatches, or less. There may be no mismatches between nucleotides
in the antisense oligomer involved in pairing and a complementary
target sequence. Preferably, mismatches do not occur at contiguous
positions. For example, in an antisense oligomer containing 3
mismatch positions, it is preferred if the mismatched positions are
separated by runs (e.g., 3, 4, 5, etc.) of contiguous nucleotides
that are complementary with nucleotides in the target sequence
[0115] The use of percent identity is a common way of defining the
number of mismatches between two nucleic acid sequences. For
example, two sequences having the same nucleobase pairing capacity
(i.e., they are fully complementary) would be considered 100%
complementary. Moreover, it should be understood that both uracil
and thymidine will bind with adenine. Consequently, two molecules
that are otherwise identical in sequence would be considered
identical, even if one had uracil at position x and the other had a
thymidine at corresponding position x. Percent identity may be
calculated over the entire length of the oligomeric compound, or
over just a portion of an oligomer. For example, the percent
identity of an antisense oligomer to a target sequence can be
calculated to determine the capacity of an oligomer comprising the
targeting sequence to bind to a nucleic acid molecule comprising
the target sequence. The sequence of an antisense oligomer of this
disclosure may be at least 80%, 85%, 90%, 95%, 97%, 98%, or 99%
complementary over its entire length to a target sequence in a
COL6A1 pre-mRNA. In one embodiment, the antisense oligomer is full
complementary to a target sequence in a target sequence in a COL6A1
pre-mRNA molecule.
[0116] Antisense oligomers of this disclosure need not be identical
to the oligomer sequences disclosed herein in order to function
similarly to the antisense oligomers described herein. Shortened
versions of antisense oligomers disclosed herein, or non-identical
versions of the antisense oligomers taught herein, fall within the
scope of this disclosure. Non-identical versions are those wherein
each base does not have 100% identity with the antisense oligomers
disclosed herein. A non-identical version can include at least one
base replaced with a different base with different pairing activity
(e.g., G can be replaced by C, A, or T). Percent identity is
calculated according to the number of bases that have identical
base pairing corresponding to the oligomer to which it is being
compared. The non-identical bases may be adjacent to each other,
dispersed throughout the oligomer, or both. For example, a 16-mer
having the same sequence as nucleobases 2-17 of a 20-mer is 80%
identical to the 20-mer. Alternatively, a 20-mer containing four
nucleobases not identical to the 20-mer is also 80% identical to
the 20-mer. A 14-mer having the same sequence as nucleobases 1-14
of an 18-mer is 78% identical to the 18-mer. Such calculations are
well within the ability of those skilled in the art. Thus,
antisense oligomers of the invention comprise oligonucleotide
sequences at least 80%, 85%, 90%, 92%, 94%, 96%, or 98% identical
to antisense oligomer sequences disclosed herein, as long as the
antisense oligomers are able to modulate splicing of alpha 1(VI)
chain-m pre-mRNA molecule.
[0117] Thus, this disclosure provides antisense oligomers targeted
to a sequence in a COL6A1 pre-mRNA molecule, wherein binding of the
antisense oligomer to the target sequence results in production of
a mature a COL6A1 mRNA molecule lacking a pseudo-exon. Binding of
the antisense oligomer to the target sequence may result in the
production of a mature a COL6A1 mRNA molecule lacking SEQ ID NO:4.
Binding of the antisense oligomer to the target sequence may result
in production of a mature a COL6A1 mRNA molecule lacking SEQ ID
NO:3. Binding of the antisense oligomer to the target sequence may
result in the production of a mature a COL6A1 mRNA molecule
comprising SEQ ID NO:13. Binding of the antisense oligomer to the
target sequence may result in the production of a mature alpha
COL6A1 mRNA molecule encoding a normal alpha 1(VI) chain protein.
Binding of the antisense oligomer to the target sequence may result
in the production of a mature COL6A1 mRNA encoding a alpha 1(VI)
chain protein comprising SEQ ID NO:14.
[0118] Antisense oligomers may be DNA molecules, RNA molecules,
synthetic nucleic acid molecules, and combinations thereof. These
antisense oligomers may also comprise a modification selected from
the group consisting of a nucleoside modification, an
internucleoside modification, a sugar modification, a
sugar-internucleoside linkage modification, and combinations
thereof. These modifications may reduce degradation by a
ribonuclease or may increase the half-life of the antisense
oligomer. The antisense oligomer may be a morpholino oligomer.
[0119] The antisense oligomer may consist of between 9 and 51
nucleosides, or between 14 and 26 nucleosides, including each
integer nucleotide length between 9 and 51 nucleosides.
[0120] As has been described, the target sequence is a sequence
which, if bound by the antisense oligomer, can modulate splicing of
exon 11 and exon 12 sequences in an alpha 1(VI) chain pre-mRNA
molecule. As such, the target sequence may be located in any region
of the alpha 1(VI) chain pre-mRNA, as long as it causes the desired
effect. Thus, the target sequence may be located in intron 11 in
the alpha 1(VI) chain pre-mRNA. The target sequence can, but need
not include, any nucleobases from exon 11 or exon 12. The target
sequence does not necessarily comprise SEQ ID NO:1 or SEQ ID NO:7.
The target sequence may be located in intron 11. The target
sequence may be located within a sequence at least 90%, 95%, 97%,
or 99% identical to SEQ ID NO:3. The target sequence may be located
within a sequence consisting of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID
NO:6.
[0121] The target sequence may be at least 90%, 95%, 97% or 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:23, SEQ ID NO:24, SEQ ID NO:25, NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, and SEQ ID NO:58. The target sequence may
comprise a sequence selected from the group consisting of SEQ ID
NO:23, SEQ ID NO:24, SEQ ID NO:25, NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:45, SEQ ID NO:46, SEQ
ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID
NO:56, SEQ ID NO:57, and SEQ ID NO:58
[0122] The antisense oligomer may comprise a sequence at least 90%,
95%, 97% or 99% identical to a sequence selected from the group
consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ
ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44.
The antisense oligomer may comprise a sequence selected from the
group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID
NO:44.
[0123] Antisense oligomers comprise a nucleic acid sequence
sufficiently complementary to a target sequence in a COL6A1
pre-mRNA molecule such that the antisense oligomer specifically
hybridizes to the target sequence resulting in normal splicing of
the COL6A1 pre-mRNA molecule. As used herein, the phrase
sufficiently complementary refers to a situation in which the
number of nucleobases in the antisense oligomer that are
complementary to the nucleobases at corresponding locations in the
target sequence is high enough that the antisense oligomer
specifically hybridizes with the target sequence. Those skilled in
the art will understand that in such a situation, the nucleic acid
sequence in the antisense oligomer and the nucleic acid sequence in
the target sequence have a high degree of complementarity. Thus,
the nucleic acid sequence in the antisense oligomer and the nucleic
acid sequence in the target sequence may be at least 90%, 95%, 97%,
99%, or 100% complementary. Hybridization of the nucleic acid
sequence in the oligomer to the target sequence may result in
production of a mature COL6A1 mRNA lacking SEQ ID NO:4.
Hybridization of the nucleic acid sequence in the oligomer to the
target sequence may result in the production of a mature COL6A1
mRNA lacking SEQ ID NO:3, SEQ ID NO:9, or SEQ ID NO:12.
Hybridization of the nucleic acid sequence in the oligomer to the
target sequence may result in the production of a mature COL6A1
mRNA comprising SEQ ID NO:13. Hybridization of the nucleic acid
sequence in the oligomer to the target sequence may result in the
production of a mature COL6A1 mRNA encoding a normal alpha 1(VI)
chain protein. Hybridization of the nucleic acid sequence in the
oligomer to the target sequence may result in the production of a
mature COL6A1 mRNA encoding an alpha 1(VI) chain protein comprising
SEQ ID NO:14.
[0124] This disclosure includes expression vectors comprising, or
encoding, an antisense oligomer of the invention. As used herein,
an "expression vector" is a nucleic acid molecule comprising a
polynucleotide sequence functionally linked to a promoter, such
that transcription of the polynucleotide sequence by a polymerase
results in production of an antisense oligomer of the invention.
Exemplary expression vectors include polynucleotide molecules,
preferably DNA molecules, that are derived, for example, from a
plasmid, bacteriophage, yeast or virus (e.g., adenovirus,
adeno-associated virus, lentivirus, retrovirus, etc.), into which a
polynucleotide can be inserted or cloned. Suitable expression
vectors are known to those skilled in the art.
[0125] This disclosure also includes pharmaceutical compositions
comprising an antisense oligomer or expression vector of the
invention. Such compositions are suitable for the therapeutic
delivery of antisense oligomers, or expression vectors, described
herein. Hence, the invention provides pharmaceutical compositions
that comprise a therapeutically-effective amount of one or more of
the antisense oligomers or expression vectors described herein,
formulated together with one or more pharmaceutically-acceptable
carriers (additives) and/or diluents. While it is possible for an
antisense oligomer or expression vector of the invention to be
administered alone, it is preferable to administer the compound as
a pharmaceutical composition.
[0126] Pharmaceutical compositions of the invention may be
specially formulated for administration in solid or liquid form,
including those adapted for the following: (1) oral administration,
for example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; (2) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (3) topical application, for
example, as a cream, ointment, or a controlled-release patch or
spray applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; (5) sublingually; (6)
ocularly; (7) transdermally; (8) inhaled into the lungs, for
example, by nebulizer or aerosol inhaler; or (9) nasally. Examples
of suitable carriers, additives and diluents are described in U.S.
Patent Publication No. 2015/0361428, which is incorporated herein
by reference in its entirety.
[0127] As previously described, the inventors have discovered that
the afore-mentioned C>T mutation results in aberrant splicing of
a COL6A1 pre-mRNA. Moreover, the inventors have described antisense
oligomers that are capable of altering splicing of the mutated
pre-mRNA, thereby causing normal splicing of the mutated COL6A1
pre-mRNA. Thus, one embodiment of the invention is a method for
restoring normal splicing of a mutated COL6A1 pre-mRNA in a cell
comprising the 189C>T mutation in its COL6A1 gene, the method
comprising contacting the cell with an antisense oligomer of the
invention.
[0128] Because the afore-mentioned aberrant splicing of the COL6A1
pre-mRNA leads to the development of neuromuscular disorders, the
compounds and methods disclosed herein for restoring normal
splicing of the mutant pre-mRNA can be used for treating
individuals at risk for, or that have been diagnosed as having,
such disorders. Thus, this disclosure provides methods for
restoring normal splicing of mutated COL6A1 pre-mRNA in an
individual having the 189C>T mutation in their COL6A1 gene,
comprising administering to the individual an antisense oligomer of
the invention. This disclosure also provides a method for treating
an individual having the 189C>T mutation in their COL6A1 gene,
comprising administering to the individual an antisense oligomer of
this disclosure. This disclosure also provides methods for treating
an individual suspected of having a neuromuscular disorder, the
method comprising administering to the individual an antisense
oligomer of the invention. This disclosure also provides methods
for treating an individual diagnosed as having a neuromuscular
disorder, comprising administering to the individual an antisense
oligomer of the invention.
[0129] As used herein, the terms individual, subject, patient, and
the like, are meant to encompass any mammal that expresses alpha
1(VI) chain protein, with a preferred mammal being a human. The
terms individual, subject, and patient by themselves do not denote
a particular age, sex, race, and the like. Thus, individuals of any
age, whether male or female, are intended to be covered by this
disclosure. Likewise, the methods of this disclosure can be applied
to any race of human, including, for example, Caucasian (white),
African-American (black), Native American, Native Hawaiian,
Hispanic, Latino, Asian, and European. Such characteristics may be
significant, and in such cases, the significant characteristic(s)
(e.g., age, sex, race, etc.) will be indicated. Additionally, the
term "individual" encompasses both human and non-human animals.
Suitable non-human animals to which antisense oligomers of the
invention may be administered include, but are not limited to
companion animals (i.e. pets), food animals, work animals, or zoo
animals. Preferred animals include, but are not limited to, cats,
dogs, horses, ferrets and other Mustelids, cattle, sheep, swine,
and rodents.
[0130] Antisense oligomers of the invention may be administered to
an individual by any suitable route of administration. Examples of
such routes include, but are not limited to, oral and parenteral
routes, (e.g., intravenous (IV), subcutaneous, intraperitoneal
(IP), and intramuscular), inhalation (e.g., nebulization and
inhalation) and transdermal delivery (e.g., topical). It is
appreciated that any methods effective to deliver an antisense
oligomer into the bloodstream of an individual are also
contemplated. For example, transdermal delivery of antisense
oligomers may be accomplished by use of a pharmaceutically
acceptable carrier adapted for topical administration. Antisense
oligomers can be administered in the absence of other molecules,
such as proteins or lipids, or they be administered in a complex
with other molecules, such as proteins or lipids. For example, the
use of cationic lipids to encapsulate antisense oligomers is
disclosed in U.S. Pat. Nos. 8,569,256, and 6,806,084, which are
incorporated herein by reference in their entirety. Similarly, the
use of peptide-linked morpholino antisense oligonucleotides is
disclosed in U.S. Patent Publication No. 2015/0238627, which is
incorporated herein by reference.
Screening Vectors
[0131] As previously described, a spontaneous mutation in intron 11
of COL6A1 results in aberrant splicing of pre-mRNA transcribed from
the gene. More specifically, substitution of the cytosine at
position 189 of intron 11 (SEQ ID NO:3) with a thymidine introduces
a new 5' donor splice site into the COL6A1 pre-mRNA. The cellular
splicing apparatus joins the newly created donor site to the exon
12 acceptor site, resulting in introduction of an in-frame
pseudo-exon into the mature mRNA. Consequently, the mature COL6A1
mRNA is 72 nucleotides longer than mature mRNA from a gene encoding
a wild-type alpha 1(VI) chain. The inventors have discovered that
this altered splicing pattern, and the resulting difference in size
between mature wild-type mRNA and mature mutant mRNA, can be used
to test compounds for their ability to cause normal splicing of
COL6A1 pre-mRNA. In particular, the inventors have constructed
vectors comprising the mutant intron 11 (i.e., intron 11 containing
189C>T) flanked by exon 11 and exon 12. The exon 11-intron
11:189C>T-exon 12 construct is functionally linked to a promoter
sequence. When the construct is introduced into a cell, the exon
11-intron 11:189C>T-exon 12 sequence is transcribed into
pre-mRNA, which is then spliced to produce the mutant mRNA
comprising the pseudo-exon. This product can be detected using, for
example, hybridization assays, or PCR primers that bind to
sequences flanking the exon 11-intron 11:189C>T-exon 12
sequence. When an antisense oligomer capable of modulating splicing
is introduced into the cell, it binds to the pre-spliced mRNA
thereby blocking the new 5' donor splice site in the intron. The
cellular splicing machinery then joins exon 11 to exon 12,
resulting in normal splicing of exon 11 and exon 12. This
normally-spliced mRNA lacks the pseudo-exon and is therefore
smaller in size than the mutant spliced mRNA. Thus, a PCR product
produced from the normally spliced mRNA will be shorter than a
product produced from the mutant spliced mRNA, using the same
primers. Such constructs and their principle of operation are shown
in FIGS. 4 and 5.
[0132] Thus, this disclosure also provides recombinant nucleic acid
molecules comprising an insert containing at least a portion of
intron 11 (e.g., comprising SEQ ID NO:6), wherein the portion of
intron 11 is flanked by at least a 3' splice acceptor site and at
least a 5' splice donor site, the insert being operationally linked
to a promoter sequence. The portion of intron 11 is flanked by at
least a portion of a COL6A1 exon 11 comprising the donor site, and
at least a portion of COL6A1 exon 12 comprising the acceptor site.
The at least a portion of exon 11 may be 5' to the portion of
intron 11. The at least a portion of exon 12 may be 3' of the
portion of intron 11. The at least a portion of exon 11 may
comprise SEQ ID NO:59. The at least a portion of exon 11 may
comprise SEQ ID NO:1. The at least a portion of exon 12 may
comprise SEQ ID NO:60. The at least a portion of exon 12 may
comprise SEQ ID NO:7. Examples of such constructs are shown in
FIGS. 4, 5 and 6.
[0133] These constructs may be used in the methods of this
disclosure to determine if a compound is capable modulating
splicing of exon 11 and exon 12 of COL6A1 pre-mRNA. These methods
include: introducing a test antisense oligomer into a cell
comprising a recombinant expression vector of the invention;
performing a first polymerase chain reaction (PCR) assay on nucleic
acid molecules obtained from the cell, using a set of primers that
bind sequences flanking the 3' splice acceptor-intron 11 portion-5'
splice donor insert; and comparing the size of the PCR product with
a PCR product produced from a second PCR assay performed on a
second cell comprising the recombinant expression vector but
lacking the test antisense oligomer, and using the same pair of
primers. If the PCR product produced from the first PCR assay is
smaller than the PCR product produced in the second PCR assay, it
may be concluded that the test antisense primer is capable of
modulating splicing of exon 11 and exon 12 of COL6A1 pre-mRNA.
[0134] Once compounds that affect COL6A1 pre-mRNA splicing (e.g.,
cause normal splicing of COL6A1 pre-mRNA) have been identified
(e.g., by using the disclosed screening vectors), the effectiveness
of such compounds can be tested in whole organisms. Thus, the
present disclosure also provides transgenic animals comprising a
Col6a1 locus comprising the disclosed c.930+189C>T mutation. In
one aspect, the transgenic animal has been engineered to comprise
the c.930+189C>T mutation, or to comprise a mutation at the
corresponding location in the animal's genome. In one aspect, the
animal is a mouse. Such a mouse can be engineered so that the mouse
gene contains the c.930+189C>T mutation, or a mutation at the
corresponding location (e.g., in the COL6A1 intron 11). In one
aspect, the animal has been engineered so that a portion of the
mouse Col6a1 locus has been replaced with the corresponding portion
from the human genome. For example, the region of the mouse Col6a1
locus spanning exon 9 to exon 14 may be replaced with the human
counterpart. In one aspect, the human counterpart comprises the
c.930+189C>T mutation. Mice may also be produced in which a
portion of the mouse Col6a1 locus has been replaced with the
corresponding portion from the genome of a human having a normal
Col6a1 locus (i.e., lacking the c.930+189C>T mutation). Also
provided are guide RNAs for producing transgenic animals of the
invention.
Kits
[0135] Also included in this disclosure are kits useful for
practicing the disclosed methods. A kit for determining the
likelihood of developing a neuromuscular disorder, in accordance
with the methods of this disclosure may include: i) reagents for
selectively detecting the presence or absence of the SNP of this
disclosure in a nucleic acid sample isolated from a biological
sample obtained from an individual tested and ii) instructions for
using the kit. Thus, such kit may be used for determining the risk
of an individual to develop a neuromuscular disorder, or to
determine the risk of an individual to develop Ullrich muscular
dystrophy. Such kit may also be used for confirming a diagnosis, or
suspected diagnosis of Ullrich muscular dystrophy in an
individual.
[0136] These kits may also contain at least some of the reagents
required to determine the presence or absence of particular alleles
of this disclosure. Reagents for these kits may include, but are
not limited to, an isolated nucleic acid, preferably a primer, a
set of primers, or an array of primers, as described elsewhere
herein. The primers may be fixed to a solid substrate. The kits may
further comprise a control target nucleic acid and primers. One
skilled in the art will, without undue experimentation, able to
select primers in accordance with the requirements of the detection
methods to be utilized. The isolated nucleic acids of the kit may
also comprise a molecular label or tag. Usually, the primer, set of
primers, or array of primers, are directed to detect the presence
or absence of at least one allele of the SNP of this
disclosure.
[0137] This disclosure also provides kits for testing compounds for
their ability to modulate splicing of COL6A1 pre-mRNA. Such kits
can comprise screening vectors of the invention, related probes,
and relates primers. Such kits may also comprise control compounds,
such as oligomers, that are known to modulate splicing COL6A1
pre-mRNA. Examples of such oligomers are disclosed herein.
[0138] This disclosure also provides kits for modulating splicing
of COL6A1 pre-mRNA, and/or treating an individual suspected of
having, or diagnosed as having a neuromuscular disorder, the kit
comprising at least one antisense oligomer of the invention or a
vector encoding at least one antisense oligomer of the invention.
The kit may also comprise instructions for using the kit, and
various reagents, such as buffers, necessary to practice the
methods of the invention. These reagents or buffers may be useful
for administering an antisense oligomer of the invention to a cell
or an individual.
[0139] The kit may also comprise any material necessary to practice
the methods of the invention, such as syringes, tubes, swabs, and
the like.
[0140] These kits may also comprise various reagents, such as
buffers, necessary to practice the methods of this disclosure.
These reagents or buffers may, for example, be useful to extract
and/or purify the nucleic from the biological sample obtained from
the individual to be tested. The kit may also comprise all the
necessary materials such as microcentrifuge tubes necessary to
practice the methods of this disclosure.
EXAMPLES
Example 1. Discovery of an Intronic Mutation in the COL6A1 Gene
[0141] RNA-sequencing (RNA-seq) and whole genome sequencing (WGS)
in undiagnosed collagen VI-like patients at the NIH identified a
new intronic mutation in COL6A1. Patients with a clinical and
biochemical presentation of collagen VI-related disorder, but for
whom no mutation had been identified by routine genetic testing,
were selected for whole-transcriptome analysis. Two muscle RNA
samples were sent for RNA-sequencing, and analyzed for variations
in gene expression and splicing events. For the two patients (US6
and US8), a new splicing event was identified in COL6A1 intron 11,
which leads to the retention of a 72-bp intronic sequence between
exons 11 and 12 (FIG. 1). This splicing event was not observed in
control samples. Genomic DNA from patient US6 was further analyzed
by WGS, and a heterozygous variant was identified adjacent to the
5' splice site of the new splicing event (FIG. 1). This variant was
absent in all control genomes sequenced. The mutation (NM_001848
c.930+189C>T) predicts the creation of a 5' donor splice site
(aggc>AGgt; Human Splicing Finder Matrices splicing site motif
strength of 50.23 vs 77.07 for C and T alleles, respectively),
likely causing the retention of the intronic sequence. The
retention of the 72-bp pseudo-exon sequence was validated by RT-PCR
in RNA samples isolated from patients' muscle biopsies and cultured
fibroblasts (FIG. 2). The retention of the pseudo-exon occurs at
the N-terminal end of the triple helical domain of alpha 1(VI)
collagen, and by its position would not affect the cysteine
residues important for monomer dimerization, nor the critical
region important for assembly. The retention of the pseudo-exon
instead predicts a dominant-negative mechanism of action for this
mutation, similar to the most common exon deletion and glycine
mutations.
Example 2. Frequency and Clinical Effect of the Intronic Mutation
in the COL6A1 Gene
[0142] The intronic COL6A1 mutation was the most common molecular
defect associated with severe COL6-RD in the NIH cohort. By
investigating the NIH cohort of molecularly unconfirmed collagen
VI-like patients, the inventors uncovered a total of 15 cases
carrying the intronic mutation (C>T) on one allele. Similarly,
additional cohorts of patients from Utah, Italy, France and the UK
were screened and identified this new intronic mutation in as many
additional patients. In the NIH cohort, the intronic mutation was
the single most common molecular defect associated with severe
COL6-RD, surpassing the COL6A3 deletion of exon 16 mutation (n=12),
also associated with a severe Ullrich phenotype (FIG. 3).
[0143] This mutation is associated with a severe phenotype, typical
of dominant-negative mutations, causing Ullrich muscular dystrophy,
although with a delayed onset. On histology, the main findings were
the increased degeneration/regeneration, increased centrally
localized nuclei. Immunohistofluorescence showed absence of
colocalization of collagen VI to the basement membrane. The
phenotype of dermal fibroblasts was fairly normal for all cases
tested, with only slight reduction of matrix deposition, and slight
intracellular retention. When parental DNA samples were available
(five or more cases total), segregation analyses showed that this
mutation was de novo in all cases. Despite lack of prominent
neonatal symptoms, all patients progressed to Ullrich congenital
muscular dystrophy (UCMD), the severe end of the COL6-RD spectrum
(wheelchair dependence beginning at 7-10 years, respiratory
insufficiency with nocturnal non-invasive ventilation started by
teenage years).
Example 3. Expression of the Intronic Mutation in the COL6A1
Gene
[0144] The intronic mutation creates a new 5' splice donor that can
be used as an alternative splicing site. Using end-point PCR and
gel quantification, the inventors found that the level of
expression of the mutant allele was lower than the expression level
from the normal allele in all individuals tested (FIG. 2). In
muscle biopsies, the expression was on average around 26% of total
COL6A1 expression (FIG. 2), whereas in cultured dermal fibroblasts,
it was on average of 9% (FIG. 2). The low levels of expression of
the mutant allele is consistent with the mild matrix dysfunction in
cultured cells, but is unexpected for a dominant-negative mutation
associated with severe clinical hallmarks.
Example 4. Expression of the Pseudo-Exon Transcripts
[0145] To verify whether the low levels of expression of the
pseudo-exon transcripts could be the result of an alternative usage
of the mutant 5' splice site, one patient (US14) was identified who
carried an exonic polymorphism in close proximity to the
pseudo-exon insertion site (r51980982 T>C in exon 15), which can
be used to track the allelic origin of transcript isoforms.
Sequencing of gel-separated RT-PCR products showed that
pseudo-exon-containing transcripts were derived solely from the T
allele of rs1980982, but that normal transcripts (i.e. excluding
the pseudo-exon insertion) originated from both the C and T
alleles, suggesting that the mutant splice donor site in intron 11
is alternatively used to include or exclude the pseudo-exon (Data
not shown; the alternative usage of the mutant splice site is
illustrated in FIG. 6).
Example 5. Effect of the Intronic Mutation on Splice Site
Selection
[0146] To gain further insight into the splicing behavior of the
intronic mutation, three different minigene constructs were
prepared that used an exon-trap vector (pET01): one containing the
intron 11 sequence (pET+Int-11), one containing exons 11 to 13
(pET+Ex-11-13), and one containing exons 10 to 13 (pET+Ex-10-13),
each in both normal and mutant versions (FIG. 4). Expression of
pET+Int-11 in different cell types did not result in the inclusion
of the pseudo-exon, whereas expression of pET+Ex-11-13 did result
in an alternative splicing event, as seen by the presence of an
additional PCR product (FIG. 5). But this splicing event was
different than the one found in the patient samples, as it used a
different 3' acceptor splice site. These results confirm that the
intronic mutation does create a donor splice site.
Example 6. Effect of Morpholino Antisense Oligonucleotides on
Pseudo-Exon Exclusion
[0147] Splice-switching oligonucleotides can suppress a pseudo-exon
inclusion. To test whether antisense oligonucleotides could be used
to exclude the COL6A1 intron 11 pseudo-exon, the inventors designed
phosphorothioate morpholino antisense oligonucleotides (PMO)
targeting different locations: either the splice acceptor site
(PMO-1, PMO-1b, PMO1-c), the splice donor site (PMO-3, PMO-3b,
PMO-3c) or within the pseudo-exon, at a predicted splicing enhancer
site (PMO-2, PMO-2b, PMO-2c, PMO-2d, PMO-2e, PMO-2f, PMO-4, PMO-5)
(FIG. 8A). PMO treatment of pET-Ex-11-13-transfected cells showed
that PMO-2 and PMO-2b were the most effective at suppressing the
pseudo-exon inclusion (FIG. 8B, FIG. 8C), whereas PMO-1 and PMO-3
had only mild effect on the splicing outcome (FIG. 8B). The target
sequences of PMO-2 and PMO-2b contain several predicted binding
sites for splicing enhancer factors SRSF2 and SRSF6.
Example 7. Effect of Morpholino Antisense Oligonucleotides on
Pseudoexon Expression and Microfibril Formation Patient-Derived
Cells
[0148] The effect of the described PMOs on patient-derived
fibroblasts was determined by first comparing pseudoexon expression
levels in patient-derived fibroblasts treated for 48 h either with
single PMOs (FIG. 9A), or with a combination of PMOs (FIG. 9B) at
various doses. Following treatment, the cells were harvested,
cellular RNA isolated and amplified, and the amplified RNA analyzed
using an allele-specific quantitative reverse transcriptase PCR
(RT-PCR) assay normalized to phosphoglycerate kinase 1 (PGK1). The
results of this analysis are shown in FIGS. 9A and 9B. The results
show that the individual PMOs caused a decrease of pseudoexon
expression (FIG. 9A). The results also show that pseudoexon
expression was lower in cells treated with combinations of PMOs
than in cells treated with an individual PMO (FIG. 9B).
[0149] Treated and untreated fibroblasts were also stained for
matrix-deposited collagen, and examined by fluorescent microscopy.
This analysis showed that deposition of collagen VI microfibrils on
the matrix was greater in PMO-treated cells, compared with cells
treated with a non-targeting PMO (PMO-neg) (FIG. 9C). Further,
rotary shadowing electron microscopy showed that microfibril length
in PMO-treated cells was greater, when compared with the length of
microfibrils in PMO-neg-treated cells. Thus, PMO treatment
increased the length of collagen VI microfibrils, as seen by the
higher number of tetramers per microfibrils following treatment
(FIG. 9D).
Example 8. Modeling of COL6A1 Intron 11+189C>T Mutation in
Mouse/Human Chimeric Reporter
[0150] A chimeric splicing reporter plasmid was prepared by cloning
the mouse genomic sequence encompassing exon 11 to exon 13, and by
replacing the intron 11 sequence with human intron 11, in presence
of the wildtype (+189C) or the mutant (+189T) genotype. A schematic
of this design in shown in FIG. 10A.
[0151] To determine if the chimeric splicing sites were recognized
in mouse cells, the reporter plasmids were transfected in murine
primary skin fibroblasts, and expression from the splicing reporter
was analyzed by reverse transcriptase PCR (RT-PCR) and sequencing.
The results, which are shown in FIG. 10B demonstrate that the
mutant splice donor was recognized within this mouse-human chimeric
genomic context. Retention of intron 12 was also found as an
unexpected splicing event.
DISCUSSION
[0152] In these Examples, the inventors have described a new
mutational mechanism for collagen VI-related disorders: an intronic
mutation in COL6A1 causing the retention of an in-frame pseudo
exon. The c.930+189C>T mutation is located in intron 11, and
creates a new 5' donor splice site that when used inserts 72 bp of
intronic sequence (24 aa) at the N-terminal part of the TH domain.
Similar to exon deletions at this location, this exon insertion is
likely to act as dominant-negative, as the mutant chain would take
part in monomer formation, but would interrupt the repeated Gly-X-Y
motif at the end of the collagenous domain. Additionally, the
mutation does not affect the critical cysteine residue, so that the
chain that carries it is likely to assemble into dimer and tetramer
molecules, which may account for its strong dominant-negative
effect.
[0153] Patient muscle biopsies showed accumulation of collagen VI
in endomysium, with absence of localization at the basement
membrane, which is consistent with dominant-negative mutations.
However, dermal fibroblasts in culture did not show the expected
reduction in collagen VI matrix deposition and increase of
retention. This can be explained by the low levels of expression of
the mutant allele in these cells, likely the result of alternative
splicing which increases the ratio of normal versus mutant chain.
This also makes the dermal fibroblasts a poor model to study this
mutation in vitro.
[0154] This COL6A1 intronic mutation, despite being common in the
patient population, was originally missed because of the low level
of expression in dermal fibroblasts. This observation emphasizes
once again the relevance of performing thorough clinical
examinations, in addition to using alternative methods of mutation
detection.
[0155] In the NIH cohort of patients, this mutation was the single
most common individual mutation identified to date for COL6-RD. It
was also identified in patients from different populations after
screening several cohorts of undiagnosed collagen VI-like patients.
This mutation manifests as a severe Ullrich muscular dystrophy but
possibly with a slightly delayed onset compared to the classic
Ullrich presentation. The reasons for a low abundance of mutant
expression being associated with severe phenotype are still
elusive. With different quantification methods (RNA-seq, gel
quantification, qRT-PCR), the inventors demonstrated that the
mutant allele is expressed at lower levels than the 1:1 expected
ratio, in dermal fibroblasts but also in RNA samples freshly
isolated from muscle. It is possible that the mutant chain has
different biochemical properties such as longer half-life or
increased stability that make them prone to accumulation, and lead
to deleterious effects over time.
[0156] The minigene assays confirmed that the mutation creates a
strong 5' splice site, although the choice of the 3' acceptor may
be tissue-dependent.
[0157] Intronic mutations leading to aberrant splicing events have
been described for other disorders including other muscular
dystrophies, although most frequently as loss-of-function mutations
causing out-of-frame intron retention. Mutations causing pseudo
exon retention offer opportunity for treatment using
splice-modulating oligomers. The inventors tested PMO antisense
oligonucleotides, and the oligonucleotide directly targeting the
mutant 5' splice site did not promote skipping, possibly because
RNA secondary structure makes it a poor choice of target. The most
efficient oligonucleotide was one targeting a potential exonic
splicing enhancer site, suggesting that trans factors facilitate
the insertion of the pseudoexon.
[0158] The foregoing description of the specific embodiments will
so fully reveal the general nature of the invention that others
can, by applying knowledge within the skill of the art, readily
modify and/or adapt for various applications such specific
embodiments, without undue experimentation, without departing from
the general concept of this disclosure. Therefore, such adaptations
and modifications are intended to be within the meaning and range
of equivalents of the disclosed embodiments, based on the teaching
and guidance presented herein. It is to be understood that the
phraseology or terminology herein is for the purpose of description
and not of limitation, such that the terminology or phraseology of
the present specification is to be interpreted by the skilled
artisan in light of the teachings and guidance.
Sequence CWU 1
1
63127DNAHomo sapiens 1ggagaaaaag ggagccgtgg ggagaag 2729PRTHomo
sapiens 2Gly Glu Lys Gly Ser Arg Gly Glu Lys1 53479DNAHomo sapiens
3gtgagtgagg ctcgacctcg gagctggtct ctccaggcgc agatgtgcca tcctggacga
60gggtgtcccc ggggatgagg acagtgtccc tgacaggaga ccacgtgtcc tgcagacccg
120ctccaccgcc cctcgccgtc ccctccatct ggaaggacaa ggacagccac
ccaggcaccc 180agcaaaggcg cctgtgtcac tttcacccca ccccagagca
ggggtccccc gggcggttac 240cctctgcgga gccgggggtc ccccgggcgg
ttaccctctg cggagccggg ggtcccccgg 300gcggttaccc tctgcagagc
ggcccctccc catcactgtc agtccccatg attctcagca 360gtgatgttgt
cccctcgggt tgggggcacc caagcccctg cctcgcgtgg gcctaagcca
420ggcttgccct gccctcccca ccccaaatac cccctcacac ccgcttcctg tctccgcag
479472DNAHomo sapiens 4acccgctcca ccgcccctcg ccgtcccctc catctggaag
gacaaggaca gccacccagg 60cacccagcaa ag 72524PRTHomo sapiens 5Thr Arg
Ser Thr Ala Pro Arg Arg Pro Leu His Leu Glu Gly Gln Gly1 5 10 15Gln
Pro Pro Arg His Pro Ala Lys 20642DNAHomo sapiens 6acccaggcac
ccagcaaagg cgcctgtgtc actttcaccc ct 42727DNAHomo sapiens
7ggctccaggg gacccaaggg ctacaag 2789PRTHomo sapiens 8Gly Ser Arg Gly
Pro Lys Gly Tyr Lys1 5924DNAHomo sapiens 9cgtggggaga agacccgctc
cacc 24108PRTHomo sapiens 10Arg Gly Glu Lys Thr Arg Ser Thr1
51124DNAHomo sapiens 11cacccagcaa agggctccag ggga 24128PRTHomo
sapiens 12His Pro Ala Lys Gly Ser Arg Gly1 51324DNAHomo sapiens
13cgtggggaga agggctccag ggga 24148PRTHomo sapiens 14Arg Gly Glu Lys
Gly Ser Arg Gly1 51525RNAArtificial sequenceSynthetic 15guggagcggg
ucugcaggac acgug 251625RNAArtificial sequenceSynthetic 16ggcuguccuu
guccucccag augga 251725RNAArtificial sequenceSynthetic 17aggcaccuuu
gcugggugcc ugggu 251825RNAArtificial sequenceSynthetic 18ugaaagugac
acaggcaccu uugcu 251923RNAArtificial sequenceSynthetic 19ggugaaagug
acacaggcaa ccu 232020RNAArtificial sequenceSynthetic 20agauggaggg
gacggcgagg 202120RNAArtificial sequenceSynthetic 21ggcuguccuu
guccuuccag 202220RNAArtificial sequenceSynthetic 22gugccugggu
ggcuguccuu 202325RNAHomo sapiens 23cacguguccu gcagacccgc uccac
252425RNAHomo sapiens 24uccaucugga aggacaagga cagcc 252525RNAHomo
sapiens 25acccaggcac ccagcaaagg ugccu 252625RNAHomo sapiens
26agcaaaggug ccugugucac uuuca 252722RNAHomo sapiens 27aggugccugu
gucacuuuca cc 222820RNAHomo sapiens 28ccucgccguc cccuccaucu
202920RNAHomo sapiens 29cuggaaggac aaggacagcc 203020RNAHomo sapiens
30aaggacagcc acccaggcac 203125DNAArtificial sequenceSynthetic
31aggacacctg gtctcctgtc aggga 253225DNAArtificial sequenceSynthetic
32gctgtccttg tccttccaga tggag 253325DNAArtificial sequenceSynthetic
33tgaaagtgac acaggcacct ttgct 253425DNAArtificial sequenceSynthetic
34gtggctgtcc ttgtccttcc agatg 253523DNAArtificial sequenceSynthetic
35ttgtccttcc agatggacgg gac 233625DNAArtificial sequenceSynthetic
36gtgcctgggt cgctgtcctt gtcct 253725DNAArtificial sequenceSynthetic
37tctgcaggac acgtggtctc ctgtc 253825DNAArtificial sequenceSynthetic
38ggtctgcagg acacgtggtc tcctg 253925DNAArtificial sequenceSynthetic
39ctgtccttgt ccttccagat ggagg 254025DNAArtificial sequenceSynthetic
40ggctgtcctt gtccttccag atgga 254125DNAArtificial sequenceSynthetic
41tggctgtcct tgtccttcca gatgg 254225DNAArtificial sequenceSynthetic
42ggtggctgtc cttgtccttc cagat 254325DNAArtificial sequenceSynthetic
43tggggtgaaa gtgacacagg cacct 254425DNAArtificial sequenceSynthetic
44ggtgaaagtg acacaggcac ctttg 254525RNAHomo sapiens 45ucccugacag
gagaccacgu guccu 254625RNAHomo sapiens 46cuccaucugg aaggacaagg
acagc 254725RNAHomo sapiens 47agcaaaggug ccugugucac uuuca
254825RNAHomo sapiens 48caucuggaag gacaaggaca gccac 254923RNAHomo
sapiens 49guccccucca ucuggaagga caa 235025RNAHomo sapiens
50aggacaagga cagccaccca ggcac 255125RNAHomo sapiens 51gacaggagac
cacguguccu gcaga 255225RNAHomo sapiens 52caggagacca cguguccugc
agacc 255325RNAHomo sapiens 53ccuccaucug gaaggacaag gacag
255425RNAHomo sapiens 54uccaucugga aggacaagga cagcc 255525RNAHomo
sapiens 55ccaucuggaa ggacaaggac agcca 255625RNAHomo sapiens
56aucuggaagg acaaggacag ccacc 255725RNAHomo sapiens 57aggugccugu
gucacuuuca cccca 255825RNAHomo sapiens 58caaaggugcc ugugucacuu
ucacc 255930DNAHomo sapiens 59agccgtgggg agaaggtgag tgaggctcga
306030DNAHomo sapiens 60ttcctgtctc cgcagggctc caggggaccc
30614246DNAHomo sapiens 61gctctcactc tggctgggag cagaaggcag
cctcggtctc tgggcggcgg cggcggccca 60ctctgccctg gccgcgctgt gtggtgaccg
caggccccag acatgagggc ggcccgtgct 120ctgctgcccc tgctgctgca
ggcctgctgg acagccgcgc aggatgagcc ggagaccccg 180agggccgtgg
ccttccagga ctgccccgtg gacctgttct ttgtgctgga cacctctgag
240agcgtggccc tgaggctgaa gccctacggg gccctcgtgg acaaagtcaa
gtccttcacc 300aagcgcttca tcgacaacct gagggacagg tactaccgct
gtgaccgaaa cctggtgtgg 360aacgcaggcg cgctgcacta cagtgacgag
gtggagatca tccaaggcct cacgcgcatg 420cctggcggcc gcgacgcact
caaaagcagc gtggacgcgg tcaagtactt tgggaagggc 480acctacaccg
actgcgctat caagaagggg ctggagcagc tcctcgtggg gggctcccac
540ctgaaggaga ataagtacct gattgtggtg accgacgggc accccctgga
gggctacaag 600gaaccctgtg gggggctgga ggatgctgtg aacgaggcca
agcacctggg cgtcaaagtc 660ttctcggtgg ccatcacacc cgaccacctg
gagccgcgtc tgagcatcat cgccacggac 720cacacgtacc ggcgcaactt
cacggcggct gactggggcc agagccgcga cgcagaggag 780gccatcagcc
agaccatcga caccatcgtg gacatgatca aaaataacgt ggagcaagtg
840tgctgctcct tcgaatgcca gcctgcaaga ggacctccgg ggctccgggg
cgaccccggc 900tttgagggag aacgaggcaa gccggggctc ccaggagaga
agggagaagc cggagatcct 960ggaagacccg gggacctcgg acctgttggg
taccagggaa tgaagggaga aaaagggagc 1020cgtggggaga agggctccag
gggacccaag ggctacaagg gagagaaggg caagcgtggc 1080atcgacgggg
tggacggcgt gaagggggag atggggtacc caggcctgcc aggctgcaag
1140ggctcgcccg ggtttgacgg cattcaagga ccccctggcc ccaagggaga
ccccggtgcc 1200tttggactga aaggagaaaa gggcgagcct ggagctgacg
gggaggcggg gagaccaggg 1260agctcgggac catctggaga cgagggccag
ccgggagagc ctgggccccc cggagagaaa 1320ggagaggcgg gcgacgaggg
gaacccagga cctgacggtg cccccgggga gcggggtggc 1380cctggagaga
gaggaccacg ggggacccca ggcacgcggg gaccaagagg agaccctggt
1440gaagctggcc cgcagggtga tcagggaaga gaaggccccg ttggtgtccc
tggagacccg 1500ggcgaggctg gccctatcgg acctaaaggc taccgaggcg
atgagggtcc cccagggtcc 1560gagggtgcca gaggagcccc aggacctgcc
ggaccccctg gagacccggg gctgatgggt 1620gaaaggggag aagacggccc
cgctggaaat ggcaccgagg gcttccccgg cttccccggg 1680tatccgggca
acaggggcgc tcccgggata aacggcacga agggctaccc cggcctcaag
1740ggggacgagg gagaagccgg ggaccccgga gacgataaca acgacattgc
accccgagga 1800gtcaaaggag caaaggggta ccggggtccc gagggccccc
agggaccccc aggacaccaa 1860ggaccgcctg ggccggacga atgcgagatt
ttggacatca tcatgaaaat gtgctcttgc 1920tgtgaatgca agtgcggccc
catcgacctc ctgttcgtgc tggacagctc agagagcatt 1980ggcctgcaga
acttcgagat tgccaaggac ttcgtcgtca aggtcatcga ccggctgagc
2040cgggacgagc tggtcaagtt cgagccaggg cagtcgtacg cgggtgtggt
gcagtacagc 2100cacagccaga tgcaggagca cgtgagcctg cgcagcccca
gcatccggaa cgtgcaggag 2160ctcaaggaag ccatcaagag cctgcagtgg
atggcgggcg gcaccttcac gggggaggcc 2220ctgcagtaca cgcgggacca
gctgctgccg cccagcccga acaaccgcat cgccctggtc 2280atcactgacg
ggcgctcaga cactcagagg gacaccacac cgctcaacgt gctctgcagc
2340cccggcatcc aggtggtctc cgtgggcatc aaagacgtgt ttgacttcat
cccaggctca 2400gaccagctca atgtcatttc ttgccaaggc ctggcaccat
cccagggccg gcccggcctc 2460tcgctggtca aggagaacta tgcagagctg
ctggaggatg ccttcctgaa gaatgtcacc 2520gcccagatct gcatagacaa
gaagtgtcca gattacacct gccccatcac gttctcctcc 2580ccggctgaca
tcaccatcct gctggacggc tccgccagcg tgggcagcca caactttgac
2640accaccaagc gcttcgccaa gcgcctggcc gagcgcttcc tcacagcggg
caggacggac 2700cccgcccacg acgtgcgggt ggcggtggtg cagtacagcg
gcacgggcca gcagcgccca 2760gagcgggcgt cgctgcagtt cctgcagaac
tacacggccc tggccagtgc cgtcgatgcc 2820atggacttta tcaacgacgc
caccgacgtc aacgatgccc tgggctatgt gacccgcttc 2880taccgcgagg
cctcgtccgg cgctgccaag aagaggctgc tgctcttctc agatggcaac
2940tcgcagggcg ccacgcccgc tgccatcgag aaggccgtgc aggaagccca
gcgggcaggc 3000atcgagatct tcgtggtggt cgtgggccgc caggtgaatg
agccccacat ccgcgtcctg 3060gtcaccggca agacggccga gtacgacgtg
gcctacggcg agagccacct gttccgtgtc 3120cccagctacc aggccctgct
ccgcggtgtc ttccaccaga cagtctccag gaaggtggcg 3180ctgggctagc
ccaccctgca cgccggcacc aaaccctgtc ctcccacccc tccccactca
3240tcactaaaca gagtaaaatg tgatgcgaat tttcccgacc aacctgattc
gctagatttt 3300ttttaaggaa aagcttggaa agccaggaca caacgctgct
gcctgctttg tgcagggtcc 3360tccggggctc agccctgagt tggcatcacc
tgcgcagggc cctctggggc tcagccctga 3420gctagtgtca cctgcacagg
gccctctgag gctcagccct gagctggcgt cacctgtgca 3480gggccctctg
gggctcagcc ctgagctggc ctcacctggg ttccccaccc cgggctctcc
3540tgccctgccc tcctgcccgc cctccctcct gcctgcgcag ctccttccct
aggcacctct 3600gtgctgcatc ccaccagcct gagcaagacg ccctctcggg
gcctgtgccg cactagcctc 3660cctctcctct gtccccatag ctggtttttc
ccaccaatcc tcacctaaca gttactttac 3720aattaaactc aaagcaagct
cttctcctca gcttggggca gccattggcc tctgtctcgt 3780tttgggaaac
caaggtcagg aggccgttgc agacataaat ctcggcgact cggccccgtc
3840tcctgagggt cctgctggtg accggcctgg accttggccc tacagccctg
gaggccgctg 3900ctgaccagca ctgaccccga cctcagagag tactcgcagg
ggcgctggct gcactcaaga 3960ccctcgagat taacggtgct aaccccgtct
gctcctccct cccgcagaga ctggggcctg 4020gactggacat gagagcccct
tggtgccaca gagggctgtg tcttactaga aacaacgcaa 4080acctctcctt
cctcagaata gtgatgtgtt cgacgtttta tcaaaggccc cctttctatg
4140ttcatgttag ttttgctcct tctgtgtttt tttctgaacc atatccatgt
tgctgacttt 4200tccaaataaa ggttttcact cctctaaaaa aaaaaaaaaa aaaaaa
4246621028PRTHomo sapiens 62Met Arg Ala Ala Arg Ala Leu Leu Pro Leu
Leu Leu Gln Ala Cys Trp1 5 10 15Thr Ala Ala Gln Asp Glu Pro Glu Thr
Pro Arg Ala Val Ala Phe Gln 20 25 30Asp Cys Pro Val Asp Leu Phe Phe
Val Leu Asp Thr Ser Glu Ser Val 35 40 45Ala Leu Arg Leu Lys Pro Tyr
Gly Ala Leu Val Asp Lys Val Lys Ser 50 55 60Phe Thr Lys Arg Phe Ile
Asp Asn Leu Arg Asp Arg Tyr Tyr Arg Cys65 70 75 80Asp Arg Asn Leu
Val Trp Asn Ala Gly Ala Leu His Tyr Ser Asp Glu 85 90 95Val Glu Ile
Ile Gln Gly Leu Thr Arg Met Pro Gly Gly Arg Asp Ala 100 105 110Leu
Lys Ser Ser Val Asp Ala Val Lys Tyr Phe Gly Lys Gly Thr Tyr 115 120
125Thr Asp Cys Ala Ile Lys Lys Gly Leu Glu Gln Leu Leu Val Gly Gly
130 135 140Ser His Leu Lys Glu Asn Lys Tyr Leu Ile Val Val Thr Asp
Gly His145 150 155 160Pro Leu Glu Gly Tyr Lys Glu Pro Cys Gly Gly
Leu Glu Asp Ala Val 165 170 175Asn Glu Ala Lys His Leu Gly Val Lys
Val Phe Ser Val Ala Ile Thr 180 185 190Pro Asp His Leu Glu Pro Arg
Leu Ser Ile Ile Ala Thr Asp His Thr 195 200 205Tyr Arg Arg Asn Phe
Thr Ala Ala Asp Trp Gly Gln Ser Arg Asp Ala 210 215 220Glu Glu Ala
Ile Ser Gln Thr Ile Asp Thr Ile Val Asp Met Ile Lys225 230 235
240Asn Asn Val Glu Gln Val Cys Cys Ser Phe Glu Cys Gln Pro Ala Arg
245 250 255Gly Pro Pro Gly Leu Arg Gly Asp Pro Gly Phe Glu Gly Glu
Arg Gly 260 265 270Lys Pro Gly Leu Pro Gly Glu Lys Gly Glu Ala Gly
Asp Pro Gly Arg 275 280 285Pro Gly Asp Leu Gly Pro Val Gly Tyr Gln
Gly Met Lys Gly Glu Lys 290 295 300Gly Ser Arg Gly Glu Lys Gly Ser
Arg Gly Pro Lys Gly Tyr Lys Gly305 310 315 320Glu Lys Gly Lys Arg
Gly Ile Asp Gly Val Asp Gly Val Lys Gly Glu 325 330 335Met Gly Tyr
Pro Gly Leu Pro Gly Cys Lys Gly Ser Pro Gly Phe Asp 340 345 350Gly
Ile Gln Gly Pro Pro Gly Pro Lys Gly Asp Pro Gly Ala Phe Gly 355 360
365Leu Lys Gly Glu Lys Gly Glu Pro Gly Ala Asp Gly Glu Ala Gly Arg
370 375 380Pro Gly Ser Ser Gly Pro Ser Gly Asp Glu Gly Gln Pro Gly
Glu Pro385 390 395 400Gly Pro Pro Gly Glu Lys Gly Glu Ala Gly Asp
Glu Gly Asn Pro Gly 405 410 415Pro Asp Gly Ala Pro Gly Glu Arg Gly
Gly Pro Gly Glu Arg Gly Pro 420 425 430Arg Gly Thr Pro Gly Thr Arg
Gly Pro Arg Gly Asp Pro Gly Glu Ala 435 440 445Gly Pro Gln Gly Asp
Gln Gly Arg Glu Gly Pro Val Gly Val Pro Gly 450 455 460Asp Pro Gly
Glu Ala Gly Pro Ile Gly Pro Lys Gly Tyr Arg Gly Asp465 470 475
480Glu Gly Pro Pro Gly Ser Glu Gly Ala Arg Gly Ala Pro Gly Pro Ala
485 490 495Gly Pro Pro Gly Asp Pro Gly Leu Met Gly Glu Arg Gly Glu
Asp Gly 500 505 510Pro Ala Gly Asn Gly Thr Glu Gly Phe Pro Gly Phe
Pro Gly Tyr Pro 515 520 525Gly Asn Arg Gly Ala Pro Gly Ile Asn Gly
Thr Lys Gly Tyr Pro Gly 530 535 540Leu Lys Gly Asp Glu Gly Glu Ala
Gly Asp Pro Gly Asp Asp Asn Asn545 550 555 560Asp Ile Ala Pro Arg
Gly Val Lys Gly Ala Lys Gly Tyr Arg Gly Pro 565 570 575Glu Gly Pro
Gln Gly Pro Pro Gly His Gln Gly Pro Pro Gly Pro Asp 580 585 590Glu
Cys Glu Ile Leu Asp Ile Ile Met Lys Met Cys Ser Cys Cys Glu 595 600
605Cys Lys Cys Gly Pro Ile Asp Leu Leu Phe Val Leu Asp Ser Ser Glu
610 615 620Ser Ile Gly Leu Gln Asn Phe Glu Ile Ala Lys Asp Phe Val
Val Lys625 630 635 640Val Ile Asp Arg Leu Ser Arg Asp Glu Leu Val
Lys Phe Glu Pro Gly 645 650 655Gln Ser Tyr Ala Gly Val Val Gln Tyr
Ser His Ser Gln Met Gln Glu 660 665 670His Val Ser Leu Arg Ser Pro
Ser Ile Arg Asn Val Gln Glu Leu Lys 675 680 685Glu Ala Ile Lys Ser
Leu Gln Trp Met Ala Gly Gly Thr Phe Thr Gly 690 695 700Glu Ala Leu
Gln Tyr Thr Arg Asp Gln Leu Leu Pro Pro Ser Pro Asn705 710 715
720Asn Arg Ile Ala Leu Val Ile Thr Asp Gly Arg Ser Asp Thr Gln Arg
725 730 735Asp Thr Thr Pro Leu Asn Val Leu Cys Ser Pro Gly Ile Gln
Val Val 740 745 750Ser Val Gly Ile Lys Asp Val Phe Asp Phe Ile Pro
Gly Ser Asp Gln 755 760 765Leu Asn Val Ile Ser Cys Gln Gly Leu Ala
Pro Ser Gln Gly Arg Pro 770 775 780Gly Leu Ser Leu Val Lys Glu Asn
Tyr Ala Glu Leu Leu Glu Asp Ala785 790 795 800Phe Leu Lys Asn Val
Thr Ala Gln Ile Cys Ile Asp Lys Lys Cys Pro 805 810 815Asp Tyr Thr
Cys Pro Ile Thr Phe Ser Ser Pro Ala Asp Ile Thr Ile 820 825 830Leu
Leu Asp Gly Ser Ala Ser Val Gly Ser His Asn Phe Asp Thr Thr 835 840
845Lys Arg Phe Ala Lys Arg Leu Ala Glu Arg Phe Leu Thr Ala Gly Arg
850 855 860Thr Asp Pro Ala His Asp Val Arg Val Ala
Val Val Gln Tyr Ser Gly865 870 875 880Thr Gly Gln Gln Arg Pro Glu
Arg Ala Ser Leu Gln Phe Leu Gln Asn 885 890 895Tyr Thr Ala Leu Ala
Ser Ala Val Asp Ala Met Asp Phe Ile Asn Asp 900 905 910Ala Thr Asp
Val Asn Asp Ala Leu Gly Tyr Val Thr Arg Phe Tyr Arg 915 920 925Glu
Ala Ser Ser Gly Ala Ala Lys Lys Arg Leu Leu Leu Phe Ser Asp 930 935
940Gly Asn Ser Gln Gly Ala Thr Pro Ala Ala Ile Glu Lys Ala Val
Gln945 950 955 960Glu Ala Gln Arg Ala Gly Ile Glu Ile Phe Val Val
Val Val Gly Arg 965 970 975Gln Val Asn Glu Pro His Ile Arg Val Leu
Val Thr Gly Lys Thr Ala 980 985 990Glu Tyr Asp Val Ala Tyr Gly Glu
Ser His Leu Phe Arg Val Pro Ser 995 1000 1005Tyr Gln Ala Leu Leu
Arg Gly Val Phe His Gln Thr Val Ser Arg 1010 1015 1020Lys Val Ala
Leu Gly1025631052PRTHomo sapiens 63Met Arg Ala Ala Arg Ala Leu Leu
Pro Leu Leu Leu Gln Ala Cys Trp1 5 10 15Thr Ala Ala Gln Asp Glu Pro
Glu Thr Pro Arg Ala Val Ala Phe Gln 20 25 30Asp Cys Pro Val Asp Leu
Phe Phe Val Leu Asp Thr Ser Glu Ser Val 35 40 45Ala Leu Arg Leu Lys
Pro Tyr Gly Ala Leu Val Asp Lys Val Lys Ser 50 55 60Phe Thr Lys Arg
Phe Ile Asp Asn Leu Arg Asp Arg Tyr Tyr Arg Cys65 70 75 80Asp Arg
Asn Leu Val Trp Asn Ala Gly Ala Leu His Tyr Ser Asp Glu 85 90 95Val
Glu Ile Ile Gln Gly Leu Thr Arg Met Pro Gly Gly Arg Asp Ala 100 105
110Leu Lys Ser Ser Val Asp Ala Val Lys Tyr Phe Gly Lys Gly Thr Tyr
115 120 125Thr Asp Cys Ala Ile Lys Lys Gly Leu Glu Gln Leu Leu Val
Gly Gly 130 135 140Ser His Leu Lys Glu Asn Lys Tyr Leu Ile Val Val
Thr Asp Gly His145 150 155 160Pro Leu Glu Gly Tyr Lys Glu Pro Cys
Gly Gly Leu Glu Asp Ala Val 165 170 175Asn Glu Ala Lys His Leu Gly
Val Lys Val Phe Ser Val Ala Ile Thr 180 185 190Pro Asp His Leu Glu
Pro Arg Leu Ser Ile Ile Ala Thr Asp His Thr 195 200 205Tyr Arg Arg
Asn Phe Thr Ala Ala Asp Trp Gly Gln Ser Arg Asp Ala 210 215 220Glu
Glu Ala Ile Ser Gln Thr Ile Asp Thr Ile Val Asp Met Ile Lys225 230
235 240Asn Asn Val Glu Gln Val Cys Cys Ser Phe Glu Cys Gln Pro Ala
Arg 245 250 255Gly Pro Pro Gly Leu Arg Gly Asp Pro Gly Phe Glu Gly
Glu Arg Gly 260 265 270Lys Pro Gly Leu Pro Gly Glu Lys Gly Glu Ala
Gly Asp Pro Gly Arg 275 280 285Pro Gly Asp Leu Gly Pro Val Gly Tyr
Gln Gly Met Lys Gly Glu Lys 290 295 300Gly Ser Arg Gly Glu Lys Thr
Arg Ser Thr Ala Pro Arg Arg Pro Leu305 310 315 320His Leu Glu Gly
Gln Gly Gln Pro Pro Arg His Pro Ala Lys Gly Ser 325 330 335Arg Gly
Pro Lys Gly Tyr Lys Gly Glu Lys Gly Lys Arg Gly Ile Asp 340 345
350Gly Val Asp Gly Val Lys Gly Glu Met Gly Tyr Pro Gly Leu Pro Gly
355 360 365Cys Lys Gly Ser Pro Gly Phe Asp Gly Ile Gln Gly Pro Pro
Gly Pro 370 375 380Lys Gly Asp Pro Gly Ala Phe Gly Leu Lys Gly Glu
Lys Gly Glu Pro385 390 395 400Gly Ala Asp Gly Glu Ala Gly Arg Pro
Gly Ser Ser Gly Pro Ser Gly 405 410 415Asp Glu Gly Gln Pro Gly Glu
Pro Gly Pro Pro Gly Glu Lys Gly Glu 420 425 430Ala Gly Asp Glu Gly
Asn Pro Gly Pro Asp Gly Ala Pro Gly Glu Arg 435 440 445Gly Gly Pro
Gly Glu Arg Gly Pro Arg Gly Thr Pro Gly Thr Arg Gly 450 455 460Pro
Arg Gly Asp Pro Gly Glu Ala Gly Pro Gln Gly Asp Gln Gly Arg465 470
475 480Glu Gly Pro Val Gly Val Pro Gly Asp Pro Gly Glu Ala Gly Pro
Ile 485 490 495Gly Pro Lys Gly Tyr Arg Gly Asp Glu Gly Pro Pro Gly
Ser Glu Gly 500 505 510Ala Arg Gly Ala Pro Gly Pro Ala Gly Pro Pro
Gly Asp Pro Gly Leu 515 520 525Met Gly Glu Arg Gly Glu Asp Gly Pro
Ala Gly Asn Gly Thr Glu Gly 530 535 540Phe Pro Gly Phe Pro Gly Tyr
Pro Gly Asn Arg Gly Ala Pro Gly Ile545 550 555 560Asn Gly Thr Lys
Gly Tyr Pro Gly Leu Lys Gly Asp Glu Gly Glu Ala 565 570 575Gly Asp
Pro Gly Asp Asp Asn Asn Asp Ile Ala Pro Arg Gly Val Lys 580 585
590Gly Ala Lys Gly Tyr Arg Gly Pro Glu Gly Pro Gln Gly Pro Pro Gly
595 600 605His Gln Gly Pro Pro Gly Pro Asp Glu Cys Glu Ile Leu Asp
Ile Ile 610 615 620Met Lys Met Cys Ser Cys Cys Glu Cys Lys Cys Gly
Pro Ile Asp Leu625 630 635 640Leu Phe Val Leu Asp Ser Ser Glu Ser
Ile Gly Leu Gln Asn Phe Glu 645 650 655Ile Ala Lys Asp Phe Val Val
Lys Val Ile Asp Arg Leu Ser Arg Asp 660 665 670Glu Leu Val Lys Phe
Glu Pro Gly Gln Ser Tyr Ala Gly Val Val Gln 675 680 685Tyr Ser His
Ser Gln Met Gln Glu His Val Ser Leu Arg Ser Pro Ser 690 695 700Ile
Arg Asn Val Gln Glu Leu Lys Glu Ala Ile Lys Ser Leu Gln Trp705 710
715 720Met Ala Gly Gly Thr Phe Thr Gly Glu Ala Leu Gln Tyr Thr Arg
Asp 725 730 735Gln Leu Leu Pro Pro Ser Pro Asn Asn Arg Ile Ala Leu
Val Ile Thr 740 745 750Asp Gly Arg Ser Asp Thr Gln Arg Asp Thr Thr
Pro Leu Asn Val Leu 755 760 765Cys Ser Pro Gly Ile Gln Val Val Ser
Val Gly Ile Lys Asp Val Phe 770 775 780Asp Phe Ile Pro Gly Ser Asp
Gln Leu Asn Val Ile Ser Cys Gln Gly785 790 795 800Leu Ala Pro Ser
Gln Gly Arg Pro Gly Leu Ser Leu Val Lys Glu Asn 805 810 815Tyr Ala
Glu Leu Leu Glu Asp Ala Phe Leu Lys Asn Val Thr Ala Gln 820 825
830Ile Cys Ile Asp Lys Lys Cys Pro Asp Tyr Thr Cys Pro Ile Thr Phe
835 840 845Ser Ser Pro Ala Asp Ile Thr Ile Leu Leu Asp Gly Ser Ala
Ser Val 850 855 860Gly Ser His Asn Phe Asp Thr Thr Lys Arg Phe Ala
Lys Arg Leu Ala865 870 875 880Glu Arg Phe Leu Thr Ala Gly Arg Thr
Asp Pro Ala His Asp Val Arg 885 890 895Val Ala Val Val Gln Tyr Ser
Gly Thr Gly Gln Gln Arg Pro Glu Arg 900 905 910Ala Ser Leu Gln Phe
Leu Gln Asn Tyr Thr Ala Leu Ala Ser Ala Val 915 920 925Asp Ala Met
Asp Phe Ile Asn Asp Ala Thr Asp Val Asn Asp Ala Leu 930 935 940Gly
Tyr Val Thr Arg Phe Tyr Arg Glu Ala Ser Ser Gly Ala Ala Lys945 950
955 960Lys Arg Leu Leu Leu Phe Ser Asp Gly Asn Ser Gln Gly Ala Thr
Pro 965 970 975Ala Ala Ile Glu Lys Ala Val Gln Glu Ala Gln Arg Ala
Gly Ile Glu 980 985 990Ile Phe Val Val Val Val Gly Arg Gln Val Asn
Glu Pro His Ile Arg 995 1000 1005Val Leu Val Thr Gly Lys Thr Ala
Glu Tyr Asp Val Ala Tyr Gly 1010 1015 1020Glu Ser His Leu Phe Arg
Val Pro Ser Tyr Gln Ala Leu Leu Arg 1025 1030 1035Gly Val Phe His
Gln Thr Val Ser Arg Lys Val Ala Leu Gly 1040 1045 1050
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